Anti-CLL-1 antibodies and immunoconjugates

ABSTRACT

The invention provides anti-CLL-1 antibodies and immunoconjugates and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a division of U.S. application Ser. No.14/852,369, filed Sep. 11, 2015, which claims priority to U.S.Provisional Application Ser. No. 62/049,876, filed on Sep. 12, 2014, thecontents of each of which is incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Sep. 2, 2015, is named P32314-1-NP_SL.txt.txt, and is40,383 bytes in size.

FIELD OF THE INVENTION

The present invention relates to anti-CLL-1 antibodies andimmunoconjugates and methods of using the same.

BACKGROUND

CLL-1 (also referred to as CLEC12A, MICL, and DCAL2), encodes a memberof the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily.Members of this family share a common protein fold and have diversefunctions, such as cell adhesion, cell-cell signaling, glycoproteinturnover, and roles in inflammation and immune response. CLL-1 has beenshown to type II transmembrane receptor comprising a single C-typelectin-like domain (which is not predicted to bind either calcium orsugar), a stalk region, a transmembrane domain and a short cytoplasmictail containing an ITIM motif. Further, CLL-1 is present on monocytesand granulocytes in normal peripheral blood and bone marrow (BM), whileabsent in nonhematological tissues. CLL-1 is also expressed on acutemyeloid leukemia (AML), myelodisplastic syndrome (MDS), and chronicmyelogenous leukemia (CML) cells. In particular, CLL-1 is a leukemiastem cell (LSC)-associated surface antigen expressed on a fraction ofCD34+CD38− AML cells in CD34 positive (CD34+) AML.

Monoclonal antibody (mAb)-based therapy has become an importanttreatment modality for cancer. Leukemia is well suited to this approachbecause of the accessibility of malignant cells in the blood, bonemarrow, spleen, and lymph nodes and the well-defined immunophenotypes ofthe various lineages and stages of hematopoietic differentiation thatpermit identification of antigenic targets. Most studies for acutemyeloid leukemia (AML) have focused on CD33. However, responses with theunconjugated anti-CD33 mAb lintuzumab have had modest single agent andactivity against AML and failed to improve patient outcomes in tworandomized trials when combined with conventional chemotherapy.

There is a need in the art for safe and effective agents that target AMLincluding CLL-1 for the diagnosis and treatment of CLL-1-associatedconditions, such as cancer. The invention fulfills that need andprovides other benefits.

SUMMARY

The invention provides anti-CLL-1 antibodies and immunoconjugates andmethods of using the same.

Provided herein are isolated monoclonal anti-CLL-1 antibodies, whereinthe antibody binds an epitope and/or binds an overlapping epitopecomprising amino acids of SEQ ID NO:49 and does not bind an epitopecomprising SEQ ID NO:50 and/or SEQ ID NO:51. In some embodiments, theanti-CLL-1 antibody binds an epitope comprising amino acids of SEQ IDNO:49. In some embodiments, the anti-CLL-1 antibody binds an epitopeconsisting or consisting essentially of the amino acids of SEQ ID NO:49.In some embodiments, the epitope is determined by hydroxyl radicalfootprinting.

Further provided herein isolated antibody that binds to CLL-1, whereinthe antibody comprises (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:45; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:7. In some embodiments,the antibody comprises HVR-H2 comprising the amino acid sequence of SEQID NO:9. In some embodiments, the antibody comprises HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:47. In some embodiments, theantibody comprises HVR-H2 comprising the amino acid sequence of SEQ IDNO:11. In some embodiments, the antibody comprises HVR-H2 comprising theamino acid sequence of SEQ ID NO:43. In some embodiments, the antibodycomprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:44.

In some embodiments, the antibody comprises: (a) a heavy chain variableregion comprising the sequence of SEQ ID NO: 33 and a light chainvariable region comprising the sequence of SEQ ID NO: 32; (b) a heavychain variable region comprising the sequence of SEQ ID NO: 34 and alight chain variable region comprising the sequence of SEQ ID NO: 32;(c) a heavy chain variable region comprising the sequence of SEQ ID NO:46 and a light chain variable region comprising the sequence of SEQ IDNO: 32; or (d) a heavy chain variable region comprising the sequence ofSEQ ID NO: 48 and a light chain variable region comprising the sequenceof SEQ ID NO: 32. In some embodiments, the antibody comprises a heavychain variable region comprising the sequence of SEQ ID NO: 48 and alight chain variable region comprising the sequence of SEQ ID NO: 32. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the sequence of SEQ ID NO: 34 and a light chain variableregion comprising the sequence of SEQ ID NO: 32.

Provided herein are also isolated antibodies that binds to CLL-1,wherein the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:20. In someembodiments, the antibody comprises (a) a heavy chain variable regioncomprising the sequence of SEQ ID NO: 38 and (b) a light chain variableregion comprising the sequence of SEQ ID NO: 37.

In some embodiments of any of the antibodies, the antibody binds torecombinant human CLL-1. In some embodiments of any of the antibodies,the antibody binds to recombinant cynomolgus monkey CLL-1. In someembodiments of any of the antibodies, the antibody binds to endogenousCLL-1 on the surface of human peripheral blood mononucleocytes (PBMCs).In some embodiments of any of the antibodies, the antibody binds toendogenous CLL-1 on the surface of cynomolgus monkey PBMCs. In someembodiments of any of the antibodies, the antibody binds to endogenousCLL-1 on the surface of a cancer cell. In some embodiments of any of theantibodies, the antibody binds to endogenous CLL-1 on the surface of anAML cancer cell. In some embodiments of any of the antibodies, theantibody binds to endogenous CLL-1 on the surface of HL-60 cells. Insome embodiments of any of the antibodies, the antibody binds toendogenous CLL-1 on the surface of EOL-1 cells. In some embodiments ofany of the antibodies, the antibody binds to CLL-1 comprising a K244Qmutation (SEQ ID NO:1 with K244Q). In some embodiments of any of theantibodies, the antibody binds an epitope and/or binds an overlappingepitope comprising amino acids of SEQ ID NO:49. In some embodiments ofany of the antibodies, the antibody does not bind an epitope comprisingSEQ ID NO:50 and/or SEQ ID NO:51. In some embodiments of any of theantibodies, the antibody competes for human CLL-1 binding with R&DSystem Clone 687317antibody. In some embodiments of any of theantibodies, the antibody binds to endogenous human CLL-1 with a Kd ofless than 15 nM, less than 10 nM, less than 7 nM, less than 5 nM, orless than 3 nM. In some embodiments of any of the antibodies, theantibody binds to recombinant human CLL-1 with a Kd of less than 10 nM,less than 7 nM, less than 5 nM, or less than 3 nM. In some embodimentsof any of the antibodies, the antibody binds to recombinant cynomolgusmonkey CLL-1 with a Kd of less than 10 nM, less than 7 nM, less than 5nM, or less than 3 nM, less than 2 nM, or less than 1 nM.

In some embodiments of any of the antibodies, the antibody comprises oneor more engineered free cysteine amino acids residues. In someembodiments, the one or more engineered free cysteine amino acidresidues is located in the light chain. In some embodiments, the one ormore engineered free cysteine amino residues in the light chaincomprises V205C according to Kabat numbering. In some embodiments, theone or more engineered free cysteine amino residues in the light chaincomprises K149C according to Kabat numbering. In some embodiments, theone or more engineered free cysteine amino acid residues is located inthe heavy chain. In some embodiments, the one or more engineered freecysteine amino residues in the heavy chain comprises A1118C according toEU numbering. In some embodiments, the one or more engineered freecysteine amino residues in the heavy chain comprises S400C according toEU numbering.

In some embodiments of any of the antibodies, the antibody is amonoclonal antibody. In some embodiments of any of the antibodies, theantibody is a human or chimeric antibody. In some embodiments of any ofthe antibodies, the antibody is an antibody fragment that binds CLL-1.In some embodiments of any of the antibodies, the antibody is an IgG1,IgG2a or IgG2b antibody.

Further provided herein are isolated nucleic acid encoding theantibodies described herein. Also provided herein are host cellcomprising the nucleic acid encoding the antibodies described herein.Provided herein are also methods of producing an antibody comprisingculturing the host cell comprising the nucleic acid encoding theantibodies described herein so that the antibody is produced.

Provided herein are immunoconjugates comprising the antibodies describedherein and a cytotoxic agent. In particular, provided herein areimmunoconjugates having the formula Ab-(L-D)p, wherein:

-   -   (a) Ab is the antibody described herein;    -   (b) L is a linker;    -   (c) D is a cytotoxic agent and the cytotoxic agent is a drug;        and    -   (d) p ranges from 1-8.

In some embodiments of any of the immunoconjugates, the cytotoxic agentis selected from a maytansinoid, a calicheamicin, apyrrolobenzodiazepine, and a nemorubicin derivative. In some embodimentsof any of the immunoconjugates, D is a pyrrolobenzodiazepine of FormulaA:

-   -   wherein the dotted lines indicate the optional presence of a        double bond between C1 and C2 or C2 and C3;    -   R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR,        ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally        further selected from halo or dihalo, wherein R^(D) is        independently selected from R, CO₂R, COR, CHO, CO₂H, and halo;    -   R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR,        NH₂, NHR, NRR′, NO₂, Me₃Sn and halo;    -   R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂,        NHR, NRR′, NO₂, Me₃Sn and halo;    -   Q is independently selected from O, S and NH;    -   R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal        cation;    -   R and R′ are each independently selected from optionally        substituted C₁₋₈ alkyl, C₃₋₈ heterocyclyl and C₅₋₂₀ aryl groups,        and optionally in relation to the group NRR′, R and R′ together        with the nitrogen atom to which they are attached form an        optionally substituted 4-, 5-, 6- or 7-membered heterocyclic        ring;    -   R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷        respectively;    -   R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by        one or more heteroatoms and/or aromatic rings that are        optionally substituted; and    -   X and X′ are independently selected from O, S and N(H).

In some embodiments of any of the immunoconjugates, D has the structure:

-   -   wherein n is 0 or 1.

In some embodiments of any of the immunoconjugates, D has a structure:

In some embodiments of any of the immunoconjugates, D is a nemorubicinderivative. In some embodiments of any of the immunoconjugates, D has astructure:

In some embodiments of any of the immunoconjugates, L is cleavable by aprotease. In some embodiments of any of the immunoconjugates, L isacid-labile. In some embodiments of any of the immunoconjugates, Lcomprises hydrazone.

In some embodiments of any of the immunoconjugates, the immunoconjugatehas a structure:

In some embodiments of any of the immunoconjugates, p ranges from 2-5.

In some embodiments, pharmaceutical formulations are provided. In someembodiments, a pharmaceutical formulation comprises an immunoconjugatedescribed herein and a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical formulation comprises an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis an anthracycline. In some embodiments, the anthracycline isdaunorubicin or idarubicin. In some embodiments, the additionaltherapeutic agent is cytarabine. In some embodiments, the additionaltherapeutic agent is cladribine. In some embodiments, the additionaltherapeutic agent is fludarabine or topotecan. In some embodiments, theadditional therapeutic agent is 5-azacytidine or decitabine.

In some embodiments, methods of treatment are provided. In someembodiments, methods of treating CLL-1-positive cancers are provided. Insome embodiments, a method of treatment comprises administering to anindividual an effective amount of an immunoconjugate described herein ora pharmaceutical formulation described herein. In some embodiments, thecancer is a cancer. In some embodiments, the cancer is acute myeloidleukemia (AML), chronic myeloid leukemia (CML), and/or myelodysplasticsyndrome (MDS). In some embodiments, the cancer is CLL-1 positive. Insome embodiments, the CLL-1-positive cancer is AML. In some embodiments,the method comprises administering an additional therapeutic agent tothe individual. In some embodiments, the additional therapeutic agent isan anthracycline. In some embodiments, the anthracycline is daunorubicinor idarubicin. In some embodiments, the additional therapeutic agent iscytarabine. In some embodiments, the additional therapeutic agent iscladribine. In some embodiments, the additional therapeutic agent isfludarabine or topotecan. In some embodiments, the additionaltherapeutic agent is 5-azacytidine or decitabine.

In some embodiments of any of the methods, the method further comprisesadministering to the subject a PD-1 axis binding antagonist or anadditional therapeutic agent. In some embodiments, the PD-1 axis bindingantagonist is a PD-1 binding antagonist. In some embodiments, the PD-1axis binding antagonist is a PD-L1 binding antagonist. In someembodiments, the PD-1 axis binding antagonist is a PD-L2 bindingantagonist.

In some embodiments, methods of inhibiting proliferation of aCLL-1-positive cell are provided. In some embodiments, the methodcomprises exposing the cell to an immunoconjugate described herein underconditions permissive for binding of the immunoconjugate to CLL-1 on thesurface of the cell, thereby inhibiting proliferation of the cell. Insome embodiments, the cell is an AML cancer cell.

In some embodiments, a method of detecting human CLL-1 in a biologicalsample is provided. In some embodiments, a method comprises contactingthe biological sample with an anti-CLL-1 antibody under conditionspermissive for binding of the anti-CLL-1 antibody to a naturallyoccurring human CLL-1, and detecting whether a complex is formed betweenthe anti-CLL-1 antibody and a naturally occurring human CLL-1 in thebiological sample. In some embodiments, an anti-CLL-1 antibody is anantibody described herein. In some embodiments, the biological sample isan AML cancer sample.

In some embodiments, a method for detecting a CLL-1-positive cancer isprovided. In some such embodiments, a method comprises (i) administeringa labeled anti-CLL-1 antibody to a subject having or suspected of havinga CLL-1-positive cancer, and (ii) detecting the labeled anti-CLL-1antibody in the subject, wherein detection of the labeled anti-CLL-1antibody indicates a CLL-1-positive cancer in the subject. In someembodiments, an anti-CLL-1 antibody is an antibody described herein. Insome such embodiments, the labeled anti-CLL-1 antibody comprises ananti-CLL-1 antibody conjugated to a positron emitter. In someembodiments, the positron emitter is ⁸⁹Zr.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B shows alignment of the light chain variable region sequences(A) and heavy chain variable region sequences (B) of murine (m) 6E7 (SEQID NOS: 30 and 31, respectively), m21C9 (SEQ ID NOS: 37 and 38,respectively), m20B1 (SEQ ID NOS: 35 and 36, respectively), and m28H12(SEQ ID NOS: 41 and 42, respectively).

FIGS. 2A-B shows alignment of the light chain variable region sequences(A) and heavy chain variable region sequences (B) of K1H1 (SEQ ID NOS:72 and 73, respectively), m6E7 (SEQ ID NOS: 30 and 31, respectively),humanized (h) 6E7.L4H1e (SEQ ID NOS: 32 and 33, respectively), andh6E7.L4H1e.A54 (SEQ ID NOS: 32 and 34, respectively).

FIGS. 3A-B shows alignment of the light chain variable region sequences(A) and heavy chain variable region sequences (B) of K1H1 (SEQ ID NOS:72 and 73, respectively), m21C9 (SEQ ID NOS: 37 and 38, respectively),and h21C9.L2H3 (SEQ ID NOS: 39 and 40, respectively).

FIG. 4 shows change in tumor volume (mm³) over time upon treatment withch21C9, ch3H10, ch28H12, ch20B1, and ch6E7 conjugated to PNU via acysteine engineered heavy chain at amino acid residue 118 according toEU numbering (A118C) at 10 μg/m² in the EOL-1 xenograft model.

FIG. 5 shows change in tumor volume (mm³) over time upon treatment withch21C9, ch3H10, ch28H12, ch20B1, and ch6E7 conjugated to PNU via acysteine engineered heavy chain at amino acid residue 118 according toEU numbering (A118C) at 10 μg/m² in the HL-60 xenograft model.

FIG. 6 shows change in tumor volume (mm³) over time upon treatment withthe humanized antibody 6E7.L4H1e or 21C9.L2H3 with a cysteine engineeredheavy chain at amino acid residue 118 according to EU numbering (A118C)or cysteine engineered light chain at amino acid residue number 149according to Kabat numbering (K149C) conjugated to PBD (SG34) at 10μg/m² or 20 μg/m² in HL-60 xenograft model. A structure of the antibodyconjugated to SG34 is shown below:

FIG. 7 shows change in tumor volume (mm³) over time upon treatment withthe humanized antibody 6E7.L4H1e or 6E7.L4H1eN54A with a cysteineengineered light chain at amino acid residue number 149 according toKabat numbering (K149C) conjugated to PBD (SG34) at 5 μg/m², 10 μg/m²,or 20 μg/m² in HL-60 xenograft model.

DETAILED DESCRIPTION

I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-CLL-1 antibody” and “an antibody that binds to CLL-1”refer to an antibody that is capable of binding CLL-1 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting CLL-1. In one embodiment, the extent ofbinding of an anti-CLL-1 antibody to an unrelated, non-CLL-1 protein isless than about 10% of the binding of the antibody to CLL-1 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to CLL-1 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤5 nm, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001nM (e.g., 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹Mto 10⁻¹³ M). In certain embodiments, an anti-CLL-1 antibody binds to anepitope of CLL-1 that is conserved among CLL-1 from different species.

The term “antibody” is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody and that bindsthe antigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia(CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia(APL), chronic myeloproliferative disorder, thrombocytic leukemia,precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursorT-cell acute lymphoblastic leukemia (preT-ALL), multiple myeloma (MM),mast cell disease, mast cell leukemia, mast cell sarcoma, myeloidsarcomas, lymphoid leukemia, and undifferentiated leukemia. In someembodiments, the cancer is myeloid leukemia. In some embodiments, thecancer is acute myeloid leukemia (AML).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds. In some embodiments, the particular site onan antigen molecule to which an antibody binds is determined by hydroxylradical footprinting.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The term “glycosylated forms of CLL-1” refers to naturally occurringforms of CLL-1 that are post-translationally modified by the addition ofcarbohydrate residues.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-CLL-1 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “CLL-1,” as used herein, refers to any native, mature CLL-1which results from processing of a CLL-1 precursor protein in a cell.The term includes CLL-1 from any vertebrate source, including mammalssuch as primates (e.g. humans and cynomolgus monkeys) and rodents (e.g.,mice and rats), unless otherwise indicated. The term also includesnaturally occurring variants of CLL-1, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human CLL-1 proteinsequence is shown in SEQ ID NO:1. In some embodiments, the human CLL-1protein sequence comprises the K244Q SNP (SEQ ID NO:1, wherein K244 isQ). The amino acid sequence of an exemplary extracellular domain is theamino acids of SEQ ID NO:2. The amino acid sequence of an exemplaryC-type lectin like domain (CTLD) is the amino acids of SEQ ID NO:3. Theamino acid sequence of an exemplary cynomolgus monkey CLL-1 protein isshown in SEQ ID NO:4.

The term “CLL-1-positive cancer” refers to a cancer comprising cellsthat express CLL-1 on their surface. In some embodiments, expression ofCLL-1 on the cell surface is determined, for example, using antibodiesto CLL-1 in a method such as immunohistochemistry, FACS, etc.Alternatively, CLL-1 mRNA expression is considered to correlate to CLL-1expression on the cell surface and can be determined by a methodselected from in situ hybridization and RT-PCR (including quantitativeRT-PCR).

The term “CLL-1-positive cell” refers to a cell that expresses CLL-1 onits surface.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

A “chemotherapeutic agent” refers to a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlomaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylomithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®,Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib),proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779;tipifamib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersensodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below);tyrosine kinase inhibitors; serine-threonine kinase inhibitors such asrapamycin (sirolimus, RAPAMUNE®); famesyltransferase inhibitors such aslonafamib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and tripterelin; sex steroids, including progestines such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretionic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

The term “immunosuppressive agent” as used herein for adjunct therapyrefers to substances that act to suppress or mask the immune system ofthe mammal being treated herein. This would include substances thatsuppress cytokine production, down-regulate or suppress self-antigenexpression, or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,glucocorticoids such as cortisol or aldosterone, anti-inflammatoryagents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor,or a leukotriene receptor antagonist; purine antagonists such asazathioprine or mycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; steroids such as corticosteroids or glucocorticosteroidsor glucocorticoid analogs, e.g., prednisone, methylprednisolone,including SOLU-MEDROL® methylprednisolone sodium succinate, anddexamethasone; dihydrofolate reductase inhibitors such as methotrexate(oral or subcutaneous); anti-malarial agents such as chloroquine andhydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokinereceptor antibodies including anti-interferon-alpha, -beta, or -gammaantibodies, antitumor necrosis factor(TNF)-alpha antibodies (infliximab(REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept),anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies andanti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptorantibodies and antagonists (such as ACTEMRA™ (tocilizumab)); anti-LFA-1antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies,preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptidecontaining a LFA-3 binding domain (WO 90/08187 published Jul. 26, 1990);streptokinase; transforming growth factor-beta (TGF-beta);streptodornase; RNA or DNA from the host; FK506; RS-61443; chlorambucil;deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No.5,114,721); T-cell receptor fragments (Offner et al., Science, 251:430-432 (1991); WO 90/11294; laneway, Nature, 341: 482 (1989); and WO91/01133); BAFF antagonists such as BAFF antibodies and BR3 antibodiesand zTNF4 antagonists (for review, see Mackay and Mackay, TrendsImmunol., 23:113-5 (2002) and see also definition below); biologicagents that interfere with T cell helper signals, such as anti-CD40receptor or anti-CD40 ligand (CD154), including blocking antibodies toCD40-CD40 ligand (e.g., Durie et al., Science, 261: 1328-30 (1993);Mohan et al., J. Immunol., 154: 1470-80 (1995)) and CTLA4-Ig (Finck etal., Science, 265: 1225-7 (1994)); and T-cell receptor antibodies (EP340,109) such as T10B9. Some preferred immunosuppressive agents hereininclude cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF,or methotrexate.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partner, so as to remove T-cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T-cell function (e.g., proliferation, cytokineproduction, target cell killing). As used herein, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to one ormore of its binding partners. In a specific aspect, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T-cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist isMDX-1106 (nivolumab) described herein. In another specific aspect, aPD-1 binding antagonist is MK-3475 (lambrolizumab) described herein. Inanother specific aspect, a PD-1 binding antagonist is CT-011(pidilizumab) described herein. In another specific aspect, a PD-1binding antagonist is AMP-224 described herein.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280Adescribed herein. In still another specific aspect, an anti-PD-L1antibody is MEDI4736 described herein.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific aspect, the PD-L2 bindingantagonist inhibits binding of PD-L2 to PD-1. In some embodiments, thePD-L2 antagonists include anti-PD-L2 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L2 witheither one or more of its binding partners, such as PD-1. In oneembodiment, a PD-L2 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L2 so as rendera dysfunctional T-cell less dysfunctional (e.g., enhancing effectorresponses to antigen recognition). In some embodiments, a PD-L2 bindingantagonist is an immunoadhesin.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₁₂ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 12carbon atoms. A C₁-C₁₂ alkyl group can be unsubstituted or substitutedwith one or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₆ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 6 carbonatoms. Representative “C₁-C₆ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -and n-hexyl; whilebranched C₁-C₆ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;unsaturated C₁-C₆ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, and -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A C₁-C₆ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

The term “C₁-C₄ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 4 carbonatoms. Representative “C₁-C₄ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl; while branched C₁-C₄ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl; unsaturated C₁-C₄ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl. A C₁-C₄ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

“Alkoxy” is an alkyl group singly bonded to an oxygen. Exemplary alkoxygroups include, but are not limited to, methoxy (—OCH₃) and ethoxy(—OCH₂CH₃). A “C₁-C₅ alkoxy” is an alkoxy group with 1 to 5 carbonatoms. Alkoxy groups may can be unsubstituted or substituted with one ormore groups, as described above for alkyl groups.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C5H7), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂). A “C₂-C₈ alkenyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond.

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH). A “C₂-C₈ alkynyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀-. Examples of a C1-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₅-C₂₀ aryl” is an aryl group with 5 to 20 carbon atoms in thecarbocyclic aromatic rings. Examples of C₅-C₂₀ aryl groups include, butare not limited to, phenyl, naphthyl and anthracenyl. A C5-C₂₀ arylgroup can be substituted or unsubstituted as described above for arylgroups. A “C₅-C₁₄ aryl” is an aryl group with 5 to 14 carbon atoms inthe carbocyclic aromatic rings. Examples of C₅-C₁₄ aryl groups include,but are not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₁₄ arylgroup can be substituted or unsubstituted as described above for arylgroups.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃,—PO₃H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR, —C(═O)SR,—C(═S)SR, —C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X isindependently a halogen: F, Cl, Br, or I; and each R is independently—H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group orprodrug moiety. Alkylene, alkenylene, and alkynylene groups as describedabove may also be similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 3 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Exemplary heterocycles are described, e.g., in Paquette, Leo A.,“Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₂₀ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. A C₃-C₂₀ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₂₀ heterocyclo” refers to a C₃-C₂₀ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide. In variousembodiments, linkers can comprise one or more amino acid residues, suchas valine, phenylalanine, lysine, and homolysine.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and l or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or l meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include, but are not limited to,acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991, or a lateredition.

II. Compositions and Methods

In one aspect, the invention is based, in part, on antibodies that bindto CLL-1 and immunoconjugates comprising such antibodies. Antibodies andimmunoconjugates of the invention are useful, e.g., for the diagnosis ortreatment of CLL-1-positive cancers.

The invention provides anti-CLL-1 antibodies and immunoconjugates andmethods of using the same.

Provided herein are isolated monoclonal anti-CLL-1 antibodies, whereinthe antibody binds an epitope and/or binds an overlapping epitopecomprising amino acids of SEQ ID NO:49 and does not bind an epitopecomprising SEQ ID NO:50 and/or SEQ ID NO:51. In some embodiments, theanti-CLL-1 antibody binds an epitope comprising amino acids of SEQ IDNO:49. In some embodiments, the anti-CLL-1 antibody binds an epitopeconsisting or consisting essentially of the amino acids of SEQ ID NO:49.In some embodiments, the epitope is determined by hydroxyl radicalfootprinting. In some embodiments, the epitiope as determined byhydroxyl radical footprinting has a ratio of [rate constant of theantigen]/[rate constant of the antigen and antibody complex] greaterthan about any of 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, or 3.0. In some embodiments, the epitiope as determined by hydroxylradical footprinting has a ratio of [rate constant of the antigen]/[rateconstant of the antigen and antibody complex] greater than about 2.0.

Hydroxyl radical footprinting may be performed as described in theExamples. For example, samples are exposed to hydroxyl radicals forintervals of 0, 10, 15, and 20 milliseconds (ms) using the X28c Beamline at the Brookhaven National Laboratory. The labeled samples may besubjected to deglycosylation using PNGase F. The samples may beprecipitated using Trichloroacetic acid in acetone, and subjected toLC-MS analysis. The samples may be then subjected to reduction andalkylation, digestion using Trypsin, followed by liquid chromatographycoupled with high-resolution mass spectrometry (LC-MS). The MS data maybe analyzed using ProtMapMS, resulting in dose response plots for eachpeptide. Results from the free antigen may be compared against each ofthe complex forms. A homology-based model of the antigen may begenerated using Swiss-Model software, and the solvent protected regionsmay be mapped for each of the three complexes. The selected ionchromatograms (SIC) may be extracted and integrated for the unoxidizedand all oxidized forms of peptide ion (with particular m/z). These peakarea values may be used to characterize reaction kinetics in the form ofdose response (DR) plots, which measure the loss of intact peptide as afunction of the hydroxyl radical exposure. The solvent protected regionsin the complex experience gradual oxidation reaction as opposed to thefree antigen, and the differences in the rate of oxidation (called rateconstant, RC) may serve to highlight the location of the epitope.

In some embodiments of any of the antibodies, the antibody binds torecombinant human CLL-1. In some embodiments of any of the antibodies,the antibody binds to recombinant cynomolgus monkey CLL-1. In someembodiments of any of the antibodies, the antibody binds to endogenousCLL-1 on the surface of human peripheral blood mononucleocytes (PBMCs).In some embodiments of any of the antibodies, the antibody binds toendogenous CLL-1 on the surface of cynomolgus monkey PBMCs. In someembodiments of any of the antibodies, the antibody binds to endogenousCLL-1 on the surface of a cancer cell. In some embodiments of any of theantibodies, the antibody binds to endogenous CLL-1 on the surface of anAML cancer cell. In some embodiments of any of the antibodies, theantibody binds to endogenous CLL-1 on the surface of HL-60 cells. Insome embodiments of any of the antibodies, the antibody binds toendogenous CLL-1 on the surface of EOL-1 cells. In some embodiments ofany of the antibodies, the antibody binds to CLL-1 comprising a K244Qmutation (SEQ ID NO:1 with K244Q). In some embodiments of any of theantibodies, the antibody binds an epitope and/or binds an overlappingepitope comprising amino acids of SEQ ID NO:49. In some embodiments ofany of the antibodies, the antibody does not bind an epitope comprisingSEQ ID NO:50 and/or SEQ ID NO:51. In some embodiments of any of theantibodies, the antibody competes for human CLL-1 binding with R&DSystem Clone 687317antibody. In some embodiments of any of theantibodies, the antibody binds to endogenous human CLL-1 with a Kd ofless than 15 nM, less than 10 nM, less than 7 nM, less than 5 nM, orless than 3 nM. In some embodiments of any of the antibodies, theantibody binds to recombinant human CLL-1 with a Kd of less than 10 nM,less than 7 nM, less than 5 nM, or less than 3 nM. In some embodimentsof any of the antibodies, the antibody binds to recombinant cynomolgusmonkey CLL-1 with a Kd of less than 10 nM, less than 7 nM, less than 5nM, or less than 3 nM, less than 2 nM, or less than 1 nM.

In some embodiments, the characteristics of the antibody are determinedas described herein in the Examples below.

Antibody 6E7 and Other Embodiments

In some embodiments, the invention provides an anti-CLL-1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:45; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In some embodiments, HVR-H2comprises the amino acid sequences of SEQ ID NO:9. In some embodiments,HVR-H2 comprises the amino acid sequences of SEQ ID NO:47. In someembodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:11.In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ IDNO:43. In some embodiments, HVR-H2 comprises the amino acid sequences ofSEQ ID NO:44.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:45; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:10. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:10. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:10 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:7. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:10, HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:45. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:45; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:10. In some embodiments, HVR-H2 comprises the amino acidsequences of SEQ ID NO:9. In some embodiments, HVR-H2 comprises theamino acid sequences of SEQ ID NO:47. In some embodiments, HVR-H2comprises the amino acid sequences of SEQ ID NO:11. In some embodiments,HVR-H2 comprises the amino acid sequences of SEQ ID NO:43. In someembodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:44.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:7. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:8, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:45, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:10; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:45; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In some embodiments, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:9; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In some embodiments, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:47; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In some embodiments, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In some embodiments, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:43; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In some embodiments, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In any of the above embodiments, an anti-CLL-1 antibody is humanized. Inone embodiment, an anti-CLL-1 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CLL-1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46and/or SEQ ID NO:48. In certain embodiments, a VH sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQID NO:46 and/or SEQ ID NO:48 contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-CLL-1 antibody comprising that sequence retainsthe ability to bind to CLL-1. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48. Incertain embodiments, a total of 1 to 5 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:31, SEQ ID NO:33, SEQID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CLL-1 antibody comprisesthe VH sequence of SEQ ID NO:31, SEQ ID NO:33, and/or SEQ ID NO:34,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:45, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:10. In someembodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:9.In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ IDNO:47. In some embodiments, HVR-H2 comprises the amino acid sequences ofSEQ ID NO:11. In some embodiments, HVR-H2 comprises the amino acidsequences of SEQ ID NO:43. In some embodiments, HVR-H2 comprises theamino acid sequences of SEQ ID NO:44.

In another aspect, an anti-CLL-1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:30 and/or SEQ ID NO:32.In certain embodiments, a VL sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequenceof SEQ ID NO:30 and/or SEQ ID NO:32 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CLL-1 antibody comprising that sequenceretains the ability to bind to CLL-1. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO:30 and/or SEQ ID NO:32. In certain embodiments, a total of 1to 5 amino acids have been substituted, inserted and/or deleted in SEQID NO:30 and/or SEQ ID NO:32. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-CLL-1 antibody comprises the VL sequence ofSEQ ID NO:30 and/or SEQ ID NO:32, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, an anti-CLL-1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:31 and SEQ ID NO:30, respectively, including post-translationalmodifications of those sequences. In one embodiment, the antibodycomprises the VH and VL sequences in SEQ ID NO:33 and SEQ ID NO:32,respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:34 and SEQ ID NO:33, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:46 and SEQID NO:33, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:48 and SEQ ID NO:33, respectively, includingpost-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CLL-1 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CLL-1 antibody comprising a VH sequence of SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48 anda VL sequence of SEQ ID NO:30 and/or SEQ ID NO:32, respectively.

Provided herein are antibodies comprising a light chain variable domaincomprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabatnumbering as depicted in FIG. 2A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 2B. In some embodiments, the antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 2A. In some embodiments, the antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 2B.

In a further aspect of the invention, an anti-CLL-1 antibody accordingto any of the above embodiments is a monoclonal antibody, including ahuman antibody. In one embodiment, an anti-CLL-1 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

Antibody 20B1 and Other Embodiments

In some embodiments, the invention provides an anti-CLL-1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:15; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:16; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:17; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:12; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:14.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:16; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:17. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:17. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:17 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:14. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:17, HVR-L3 comprising the amino acid sequence of SEQ ID NO:14, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:16. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 15; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:16; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:17.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:14. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:12; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:13; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:14.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:15, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:16, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:17; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:12, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:13, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:14.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:15; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 16; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:17; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:12; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:14. In some embodiments,the antibody comprises (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 15; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:16; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:17;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:13; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In any of the above embodiments, an anti-CLL-1 antibody is humanized. Inone embodiment, an anti-CLL-1 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CLL-1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:36 and SEQ ID NO:35, respectively, including post-translationalmodifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CLL-1 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CLL-1 antibody comprising a VH sequence of SEQ IDNO:36 and a VL sequence of SEQ ID NO:35, respectively.

Provided herein are antibodies comprising a light chain variable domaincomprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabatnumbering as depicted in FIG. 1A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 1B.

In a further aspect of the invention, an anti-CLL-1 antibody accordingto any of the above embodiments is a monoclonal antibody, including ahuman antibody. In one embodiment, an anti-CLL-1 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

Antibody 21C9 and Other Embodiments

In some embodiments, the invention provides an anti-CLL-1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:18; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:20.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:22; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:23. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:23. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:23 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:20. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:23, HVR-L3 comprising the amino acid sequence of SEQ ID NO:20, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:22. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:22; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:23.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 18; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:19; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:20. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:18; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:19; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:20.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:21, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:22, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:23; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:18, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:19, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:20.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:18; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:20. In some embodiments,the antibody comprises (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 18; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:20.

In any of the above embodiments, an anti-CLL-1 antibody is humanized. Inone embodiment, an anti-CLL-1 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CLL-1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:38 and/or SEQ ID NO:40. In certain embodiments, aVH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identity to the amino acid sequence of SEQ ID NO:38 and/or SEQ IDNO:40 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CLL-1 antibody comprising that sequence retains the ability to bindto CLL-1. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:38 and/or SEQ IDNO:40. In certain embodiments, a total of 1 to 5 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:38 and/or SEQ IDNO:40. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-CLL-1 antibody comprises the VH sequence of SEQ ID NO:38 and/or SEQID NO:40, including post-translational modifications of that sequence.In a particular embodiment, the VH comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:21, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22,and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23.

In another aspect, an anti-CLL-1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:37 and/or SEQ ID NO:39.In certain embodiments, a VL sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequenceof SEQ ID NO:37 and/or SEQ ID NO:39 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CLL-1 antibody comprising that sequenceretains the ability to bind to CLL-1. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO:37 and/or SEQ ID NO:39. In certain embodiments, a total of 1to 5 amino acids have been substituted, inserted and/or deleted in SEQID NO:37 and/or SEQ ID NO:39. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-CLL-1 antibody comprises the VL sequence ofSEQ ID NO:37 and/or SEQ ID NO:39, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:18; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:19; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:20.

In another aspect, an anti-CLL-1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:38 and SEQ ID NO:37, respectively, including post-translationalmodifications of those sequences. In one embodiment, the antibodycomprises the VH and VL sequences in SEQ ID NO:40 and SEQ ID NO:39,respectively, including post-translational modifications of thosesequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CLL-1 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CLL-1 antibody comprising a VH sequence of SEQ IDNO:38 and/or SEQ ID NO:40 and a VL sequence of SEQ ID NO:37 and/or SEQID NO:39, respectively.

Provided herein are antibodies comprising a light chain variable domaincomprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabatnumbering as depicted in FIG. 3A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 3B. In some embodiments, the antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 3A. In some embodiments, the antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 3B.

In a further aspect of the invention, an anti-CLL-1 antibody accordingto any of the above embodiments is a monoclonal antibody, including ahuman antibody. In one embodiment, an anti-CLL-1 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

Antibody 28H12 and Other Embodiments

In some embodiments, the invention provides an anti-CLL-1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:27; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:28; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:29; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:24; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:25; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:26.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:27; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:28; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:29. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:29. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:29 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:26. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:29, HVR-L3 comprising the amino acid sequence of SEQ ID NO:26, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:28. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:27; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:28; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:29.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:24; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:25; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:26. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:24; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:25; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:26.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:27, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:28, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:29; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:24, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:25, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:26.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:27; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:28; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:29; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:24; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:25; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:26. In some embodiments,the antibody comprises (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:27; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:28; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:29;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:24; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:25; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.

In any of the above embodiments, an anti-CLL-1 antibody is humanized. Inone embodiment, an anti-CLL-1 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CLL-1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:42 and SEQ ID NO:41, respectively, including post-translationalmodifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CLL-1 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CLL-1 antibody comprising a VH sequence of SEQ IDNO:42 and a VL sequence of SEQ ID NO:41, respectively.

Provided herein are antibodies comprising a light chain variable domaincomprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabatnumbering as depicted in FIG. 1A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 1B.

In a further aspect of the invention, an anti-CLL-1 antibody accordingto any of the above embodiments is a monoclonal antibody, including ahuman antibody. In one embodiment, an anti-CLL-1 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CLL-1 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM, and optionally is ≥10⁻¹³ M. (e.g. 10⁻⁸M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20 ™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000, BAICORE®-T200 or a BIACORE®-3000(BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5chips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE®, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) and/or HBS-P (0.01 M Hepes pH7.4, 0.15M NaCl, 0.005% Surfactant P20)before injection at a flow rate of 5 μl/minute and/or 30 μl/minute toachieve approximately 10 response units (RU) of coupled protein.Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for CLL-1 and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for CLL-1 andthe other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofCLL-1. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express CLL-1. Bispecific antibodies can beprepared as full length antibodies or antibody fragments. In someembodiments, multispecific antibodies are monoclonal antibodies thathave binding specificities for at least two different sites.

In some embodiments, multispecific antibodies are monoclonal antibodiesthat have binding specificities for at least two different antigenbinding sites (such as a bispecific antibody). In some embodiments, thefirst antigen-binding domain and the second antigen-binding domain ofthe multispecific antibody may bind the two epitopes within one and thesame molecule (intramolecular binding). For example, the firstantigen-binding domain and the second antigen-binding domain of themultispecific antibody may bind to two different epitopes on the sameCLL-1 molecule. In certain embodiments, the two different epitopes thata multispecific antibody binds are epitopes that are not normally boundat the same time by one monospecific antibody, such as e.g. aconventional antibody or one immunoglobulin single variable domain. Insome embodiments, the first antigen-binding domain and the secondantigen-binding domain of the multispecific antibody may bind epitopeslocated within two distinct molecules (intermolecular binding). Forexample, the first antigen-binding domain of the multispecific antibodymay bind to one epitope on one CLL-1 molecule, whereas the secondantigen-binding domain of the multispecific antibody may bind to anotherepitope on a different CLL-1 molecule, thereby cross-linking the twomolecules.

In some embodiments, the antigen-binding domain of a multispecificantibody (such as a bispecific antibody) comprises two VH/VL units,wherein a first VH/VL unit binds to a first epitope and a second VH/VLunit binds to a second epitope, wherein each VH/VL unit comprises aheavy chain variable domain (VH) and a light chain variable domain (VL).Such multispecific antibodies include, but are not limited to, fulllength antibodies, antibodies having two or more VL and VH domains, andantibody fragments (such as Fab, Fv, dsFv, scFv, diabodies, bispecificdiabodies and triabodies, antibody fragments that have been linkedcovalently or non-covalently). A VH/VL unit that further comprises atleast a portion of a heavy chain variable region and/or at least aportion of a light chain variable region may also be referred to as an“arm” or “hemimer” or “half antibody.” In some embodiments, a hemimercomprises a sufficient portion of a heavy chain variable region to allowintramolecular disulfide bonds to be formed with a second hemimer. Insome embodiments, a hemimer comprises a knob mutation or a holemutation, for example, to allow heterodimerization with a second hemimeror half antibody that comprises a complementary hole mutation or knobmutation. Knob mutations and hole mutations are discussed further below.

In certain embodiments, a multispecific antibody provided herein may bea bispecific antibody. The term “bispecific antibody” is as used hereinrefers to a multispecific antibody comprising an antigen-binding domainthat is capable of binding to two different epitopes on one molecule oris capable of binding to epitopes on two different molecules. Abispecific antibody may also be referred to herein as having “dualspecificity” or as being “dual specific.” Exemplary bispecificantibodies may bind both CLL-1 and any other antigen. In certainembodiments, one of the binding specificities is for CLL-1 and the otheris for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certain embodiments,bispecific antibodies may bind to two different epitopes of the sameCLL-1 molecule. In certain embodiments, bispecific antibodies may bindto two different epitopes on two different CLL-1 molecules. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress CLL-1. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168), WO2009/089004, US2009/0182127, US2011/0287009,Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, andKontermann (2005) Acta Pharmacol. Sin., 26:1-9). The term“knob-into-hole” or “KnH” technology as used herein refers to thetechnology directing the pairing of two polypeptides together in vitroor in vivo by introducing a protuberance (knob) into one polypeptide anda cavity (hole) into the other polypeptide at an interface in which theyinteract. For example, KnHs have been introduced in the Fc:Fc bindinginterfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies (see,e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, andZhu et al., 1997, Protein Science 6:781-788). In some embodiments, KnHsdrive the pairing of two different heavy chains together during themanufacture of multispecific antibodies. For example, multispecificantibodies having KnH in their Fc regions can further comprise singlevariable domains linked to each Fc region, or further comprise differentheavy chain variable domains that pair with similar or different lightchain variable domains. KnH technology can be also be used to pair twodifferent receptor extracellular domains together or any otherpolypeptide sequences that comprises different target recognitionsequences (e.g., including affibodies, peptibodies and other Fcfusions).

The term “knob mutation” as used herein refers to a mutation thatintroduces a protuberance (knob) into a polypeptide at an interface inwhich the polypeptide interacts with another polypeptide. In someembodiments, the other polypeptide has a hole mutation.

The term “hole mutation” as used herein refers to a mutation thatintroduces a cavity (hole) into a polypeptide at an interface in whichthe polypeptide interacts with another polypeptide. In some embodiments,the other polypeptide has a knob mutation.

A brief nonlimiting discussion is provided below.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g., by alteringnucleic acid encoding the interface). In some embodiments, nucleic acidencoding the interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The side chain volumes of thevarious amino residues are shown, for example, in Table 1 ofUS2011/0287009. A mutation to introduce a “protuberance” may be referredto as a “knob mutation.”

In some embodiments, import residues for the formation of a protuberanceare naturally occurring amino acid residues selected from arginine (R),phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments,an import residue is tryptophan or tyrosine. In some embodiment, theoriginal residue for the formation of the protuberance has a small sidechain volume, such as alanine, asparagine, aspartic acid, glycine,serine, threonine or valine.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g. by altering nucleic acid encoding theinterface). In some embodiments, nucleic acid encoding the interface ofthe second polypeptide is altered to encode the cavity. To achieve this,the nucleic acid encoding at least one “original” amino acid residue inthe interface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue. Insome embodiments, import residues for the formation of a cavity arenaturally occurring amino acid residues selected from alanine (A),serine (S), threonine (T) and valine (V). In some embodiments, an importresidue is serine, alanine or threonine. In some embodiments, theoriginal residue for the formation of the cavity has a large side chainvolume, such as tyrosine, arginine, phenylalanine or tryptophan. Amutation to introduce a “cavity” may be referred to as a “holemutation.”

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity may, in some instances, rely on modeling the protuberance/cavitypair based upon a three-dimensional structure such as that obtained byX-ray crystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

In some embodiments, a knob mutation in an IgG1 constant region is T366W(EU numbering). In some embodiments, a hole mutation in an IgG1 constantregion comprises one or more mutations selected from T366S, L368A andY407V (EU numbering). In some embodiments, a hole mutation in an IgG1constant region comprises T366S, L368A and Y407V (EU numbering).

In some embodiments, a knob mutation in an IgG4 constant region is T366W(EU numbering). In some embodiments, a hole mutation in an IgG4 constantregion comprises one or more mutations selected from T366S, L368A, andY407V (EU numbering). In some embodiments, a hole mutation in an IgG4constant region comprises T366S, L368A, and Y407V (EU numbering).

Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (see,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber etal., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodiesas described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies” or “dual-variable domainimmunoglobulins” (DVDs) are also included herein (see, e.g., US2006/0025576A1, and Wu et al. Nature Biotechnology (2007)).). Theantibody or fragment herein also includes a “Dual Acting FAb” or “DAF”comprising an antigen binding site that binds to CLL-1 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH₂ domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fe Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

In some embodiments, one or more amino acid modifications may beintroduced into the Fc portion of the antibody provided herein in orderto increase IgG binding to the neonatal Fc receptor. In certainembodiments, the antibody comprises the following three mutationsaccording to EU numbering: M252Y, S254T, and T256E (the “YTE mutation”)(U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal ofBiological Chemistry 281(33):23514-23524 (2006). In certain embodiments,the YTE mutation does not affect the ability of the antibody to bind toits cognate antigen. In certain embodiments, the YTE mutation increasesthe antibody's serum half-life compared to the native (i.e., non-YTEmutant) antibody. In some embodiments, the YTE mutation increases theserum half-life of the antibody by 3-fold compared to the native (i.e.,non-YTE mutant) antibody. In some embodiments, the YTE mutationincreases the serum half-life of the antibody by 2-fold compared to thenative (i.e., non-YTE mutant) antibody. In some embodiments, the YTEmutation increases the serum half-life of the antibody by 4-foldcompared to the native (i.e., non-YTE mutant) antibody. In someembodiments, the YTE mutation increases the serum half-life of theantibody by at least 5-fold compared to the native (i.e., non-YTEmutant) antibody. In some embodiments, the YTE mutation increases theserum half-life of the antibody by at least 10-fold compared to thenative (i.e., non-YTE mutant) antibody. See, e.g., U.S. Pat. No.8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry281(33):23514-23524 (2006).

In certain embodiments, the YTE mutant provides a means to modulateantibody-dependent cell-mediated cytotoxicity (ADCC) activity of theantibody. In certain embodiments, the YTEO mutant provides a means tomodulate ADCC activity of a humanized IgG antibody directed against ahuman antigen. See, e.g., U.S. Pat. No. 8,697,650; see also Dall'Acquaet al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

In certain embodiments, the YTE mutant allows the simultaneousmodulation of serum half-life, tissue distribution, and antibodyactivity (e.g., the ADCC activity of an IgG antibody). See, e.g., U.S.Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of BiologicalChemistry 281(33):23514-23524 (2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 according to EU numbering (U.S. Pat. No. 6,737,056).Such Fc mutants include Fc mutants with substitutions at two or more ofamino acid positions 265, 269, 270, 297 and 327 according to EUnumbering, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine according to EU numbering (i.e., D265Aand N297A according to EU numbering) (U.S. Pat. No. 7,332,581). Incertain embodiments the Fc mutant comprises the following two amino acidsubstitutions: D265A and N297A. In certain embodiments the Fc mutantconsists of the following two amino acid substitutions: D265A and N297A.

In certain embodiments, the proline at position329 (EU numbering) (P329)of a wild-type human Fc region is substituted with glycine or arginineor an amino acid residue large enough to destroy the proline sandwichwithin the Fc/Fcγ receptor interface, that is formed between the P329 ofthe Fc and tryptophane residues W87 and W110 of FcgRIII (Sondermann etal.: Nature 406, 267-273 (20 Jul. 2000)). In a further embodiment, atleast one further amino acid substitution in the Fc variant is S228P,E233P, L234A, L235A, L235E, N297A, N₂₉₇D, or P331S and still in anotherembodiment said at least one further amino acid substitution is L234Aand L235A of the human IgG1 Fc region or S228P and L235E of the humanIgG4 Fc region, all according to EU numbering (U.S. Pat. No. 8,969,526which is incorporated by reference in its entirety).

In certain embodiments, a polypeptide comprises the Fc variant of awild-type human IgG Fc region wherein the polypeptide has P329 of thehuman IgG Fc region substituted with glycine and wherein the Fc variantcomprises at least two further amino acid substitutions at L234A andL235A of the human IgG1 Fc region or S228P and L235E of the human IgG4Fc region, and wherein the residues are numbered according to the EUnumbering (U.S. Pat. No. 8,969,526 which is incorporated by reference inits entirety). In certain embodiments, the polypeptide comprising theP329G, L234A and L235A (EU numbering) substitutions exhibit a reducedaffinity to the human FcγRIIIA and FcγRIIA, for down-modulation of ADCCto at least 20% of the ADCC induced by the polypeptide comprising thewildtype human IgG Fc region, and/or for down-modulation of ADCP (U.S.Pat. No. 8,969,526 which is incorporated by reference in its entirety).

In a specific embodiment the polypeptide comprising an Fc variant of awildtype human Fc polypeptide comprises a triple mutation: an amino acidsubstitution at position Pro329, a L234A and a L235A mutation accordingto EU numbering (P329/LALA) (U.S. Pat. No. 8,969,526 which isincorporated by reference in its entirety). In specific embodiments, thepolypeptide comprises the following amino acid substitutions: P329G,L234A, and L235A according to EU numbering.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering).

In some embodiments, alterations are made in the Fc region that resultin altered (i. e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826)according to EU numbering. See also Duncan & Winter, Nature 322:738-40(1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “THIOMAB™ antibody,” in which one or moreresidues of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drug moieties or linker-drug intermediates, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A140 (EU numbering)of the heavy chain; L174 (EU numbering) of the heavy chain; Y373 (EUnumbering) of the heavy chain; K149 (Kabat numbering) of the lightchain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering)of the heavy chain Fc region. In specific embodiments, the antibodiesdescribed herein comprise the HC-A140C (EU numbering) cysteinesubstitution. In specific embodiments, the antibodies described hereincomprise the LC-K149C (Kabat numbering) cysteine substitution. Inspecific embodiments, the antibodies described herein comprise theHC-A118C (EU numbering) cysteine substitution. Cysteine engineeredantibodies may be generated as described, e.g., in U.S. Pat. No.7,521,541.

In certain embodiments, the antibody comprises one of the followingheavy chain cysteine substitutions:

Chain EU Mutation Kabat Mutation (HC/LC) Residue Site # Site # HC T 114110 HC A 140 136 HC L 174 170 HC L 179 175 HC T 187 183 HC T 209 205 HCV 262 258 HC G 371 367 HC Y 373 369 HC E 382 378 HC S 424 420 HC N 434430 HC Q 438 434

In certain embodiments, the antibody comprises one of the followinglight chain cysteine substitutions:

Chain EU Mutation Kabat Mutation (HC/LC) Residue Site # Site # LC I 106106 LC R 108 108 LC R 142 142 LC K 149 149

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-CLL-1 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-CLL-1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-CLL-1 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells;and FS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-CLL-1 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, BIACore®, FACS,or Western blot.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies described herein forbinding to CLL-1. In certain embodiments, such a competing antibodybinds to the same epitope (e.g., a linear or a conformational epitope)that is bound by an antibody described herein. Detailed exemplarymethods for mapping an epitope to which an antibody binds are providedin Morris (1996) “Epitope Mapping Protocols,” in Methods in MolecularBiology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized CLL-1 is incubated in asolution comprising a first labeled antibody that binds to CLL-1 (e.g.,any of the antibodies described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to CLL-1. The second antibody may be present in a hybridomasupernatant. As a control, immobilized CLL-1 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to CLL-1, excess unbound antibody is removed, and theamount of label associated with immobilized CLL-1 is measured. If theamount of label associated with immobilized CLL-1 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to CLL-1. See Harlow and Lane (1988)Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CLL-1antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes(i.e., a radioconjugate).

Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and, in some embodiments intracellular accumulation therein,where systemic administration of unconjugated drugs may result inunacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates (ADC) are targeted chemotherapeutic moleculeswhich combine properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells(Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), therebyenhancing the therapeutic index by maximizing efficacy and minimizingoff-target toxicity (Carter, P. J. and Senter P. D. (2008) The CancerJour. 14(3):154-169; Chari, R. V. (2008) Ace. Chem. Res. 41:98-107

The ADC compounds of the invention include those with anticanceractivity. In some embodiments, the ADC compounds include an antibodyconjugated, i.e. covalently attached, to the drug moiety. In someembodiments, the antibody is covalently attached to the drug moietythrough a linker. The antibody-drug conjugates (ADC) of the inventionselectively deliver an effective dose of a drug to tumor tissue wherebygreater selectivity, i.e. a lower efficacious dose, may be achievedwhile increasing the therapeutic index (“therapeutic window”).

The drug moiety (D) of the antibody-drug conjugates (ADC) may includeany compound, moiety or group that has a cytotoxic or cytostatic effect.Drug moieties may impart their cytotoxic and cytostatic effects bymechanisms including but not limited to tubulin binding, DNA binding orintercalation, and inhibition of RNA polymerase, protein synthesis,and/or topoisomerase. Exemplary drug moieties include, but are notlimited to, a maytansinoid, dolastatin, auristatin, calicheamicin,pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives,PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane,trichothecene, CC1065, camptothecin, elinafide, and stereoisomers,isosteres, analogs, and derivatives thereof that have cytotoxicactivity. Nonlimiting examples of such immunoconjugates are discussed infurther detail below.

1. Exemplary Antibody-Drug Conjugates

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises an antibody (Ab) which targets a tumor cell, a drug moiety(D), and a linker moiety (L) that attaches Ab to D. In some embodiments,the antibody is attached to the linker moiety (L) through one or moreamino acid residues, such as lysine and/or cysteine.

An exemplary ADC has Formula I:Ab-(L-D)_(p)  Iwhere p is 1 to about 20. In some embodiments, the number of drugmoieties that can be conjugated to an antibody is limited by the numberof free cysteine residues. In some embodiments, free cysteine residuesare introduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary ADC of Formula I include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to a drug.In some embodiments, an antibody is exposed to reducing conditions priorto conjugation of the antibody in order to generate one or more freecysteine residues.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-drug conjugate (ADC) of Formula I. In some embodiments,antibody-drug conjugates (ADC) can be prepared using a Linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, in some embodiments, a cysteine thiol of anantibody (Ab) can form a bond with a reactive functional group of alinker or a drug-linker intermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linker components are knownin the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II:-A_(a)-W_(w)—Y_(y)  II

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2; and Ab, D, and p are defined as above forFormula I. Exemplary embodiments of such linkers are described in U.S.Pat. No. 7,498,298, which is expressly incorporated herein by reference.

In some embodiments, a linker component comprises a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, the linker may be a peptidomimetic linker such asthose described in WO2015/095227, WO2015/095124 or WO2015/095223, whichdocuments are hereby incorporated by reference in their entirety.

In some embodiments, a linker component comprises an “amino acid unit”.In some such embodiments, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline. Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In some embodiments, a linker component comprises a “spacer” unit thatlinks the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit isp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or -cyno;m is an integer ranging from 0 to 4; and p ranges from 1 to about 20. Insome embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e. loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula I:

Further nonlimiting exemplary ADCs include the structures:

where X is:

Y is:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I. Insome such embodiments, the antibody comprises more than one (linkerportion)^(a) substituents, such that more than one drug is coupled tothe antibody in the ADC of Formula I.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following linker reagents:bis-maleimido-trioxyethylene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(ε-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Exemplary Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties areknown in the art and can be isolated from natural sources according toknown methods or produced using genetic engineering techniques (see,e.g., Yu et al (2002) PNAS 99:7968-7973). Maytansinoids may also beprepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to,those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat.No. 4,256,746) (prepared, for example, by lithium aluminum hydridereduction of ansamytocin P2); C-20-hydroxy (orC-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared, for example, by demethylation using Streptomyces orActinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared,for example, by acylation using acyl chlorides), and those havingmodifications at other positions of the aromatic ring.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, forexample, by the reaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH₂ OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy(U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion ofmaytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and4,315,929) (for example, isolated from Trewia nudlflora);C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, forexample, by the demethylation of maytansinol by Streptomyces); and4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as the linkageposition. For example, an ester linkage may be formed by reaction with ahydroxyl group using conventional coupling techniques. In someembodiments, the reaction may occur at the C-3 position having ahydroxyl group, the C-14 position modified with hydroxymethyl, the C-15position modified with a hydroxyl group, and the C-20 position having ahydroxyl group. In some embodiments, the linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid drug moiety to a linker of an ADC. Each R mayindependently be H or a C₁-C₆ alkyl. The alkylene chain attaching theamide group to the sulfur atom may be methanyl, ethanyl, or propyl,i.e., m is 1, 2, or 3 (US 633410; U.S. Pat. No. 5,208,020; Chari et al(1992) Cancer Res. 52:127-131; Liu et al (1996) Proc. Natl. Acad. SciUSA 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe ADC of the invention, i.e. any combination of R and S configurationsat the chiral carbons (U.S. Pat. Nos. 7,276,497; 6,913,748; 6,441,163;US 633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al (2006) J.Med. Chem. 49:4392-4408, which are incorporated by reference in theirentirety). In some embodiments, the maytansinoid drug moiety has thefollowing stereochemistry:

Exemplary embodiments of maytansinoid drug moieties include, but are notlimited to, DM1; DM3; and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate.

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations (wherein Ab is antibody and p is 1 to about20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is1 to 4):

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20. In someembodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0425 235 B1, the disclosures of which are hereby expressly incorporatedby reference. See also Liu et al. Proc. Natl. Acad. Sci. USA93:8618-8623 (1996); and Chari et al. Cancer Research 52:127-131 (1992).

In some embodiments, antibody-maytansinoid conjugates may be prepared bychemically linking an antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (thedisclosure of which is hereby expressly incorporated by reference). Insome embodiments, ADC with an average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody. In some instances, even one molecule oftoxin/antibody is expected to enhance cytotoxicity over the use of nakedantibody.

Exemplary linking groups for making antibody-maytansinoid conjugatesinclude, for example, those described herein and those disclosed in U.S.Pat. No. 5,208,020; EP Patent 0 425 235 B1; Chari et al. Cancer Research52:127-131 (1992); US 2005/0276812 A1; and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.

(2) Auristatins and Dolastatins

Drug moieties include dolastatins, auristatins, and analogs andderivatives thereof (U.S. Pat. Nos. 5,635,483; 5,780,58; 5,767,237;6,124,431). Auristatins are derivatives of the marine mollusk compounddolastatin-10. While not intending to be bound by any particular theory,dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin/auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172; Doronina et al (2003) NatureBiotechnology 21(7):778-784; Francisco et al (2003) Blood 102(4):1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties D_(E) and D_(F), disclosed in U.S.Pat. Nos. 7,498,298 and 7,659,241, the disclosures of which areexpressly incorporated by reference in their entirety:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

R² is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹ is selected from H and C₁-C₈ alkyl;

R¹⁰ is selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(F) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Nonlimiting exemplary embodiments of ADC of Formula I comprising MMAE orMMAF and various linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Nonlimiting exemplary embodiments of ADCs of Formula I comprising MMAFand various linker components further include Ab-MC-PAB-MMAF andAb-PAB-MMAF. Immunoconjugates comprising MMAF attached to an antibody bya linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker (Doronina et al.(2006) Bioconjugate Chem. 17:114-124). In some such embodiments, drugrelease is believed to be effected by antibody degradation in the cell.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to a liquidphase synthesis method (see, e.g., E. Schröder and K. Lübke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press).Auristatin/dolastatin drug moieties may, in some embodiments, beprepared according to the methods of: U.S. Pat. No. 7,498,298; U.S. Pat.No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In some embodiments, auristatin/dolastatin drug moieties of formulasD_(E) such as MMAE, and D_(F), such as MMAF, and drug-linkerintermediates and derivatives thereof, such as MC-MMAF, MC-MMAE,MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methodsdescribed in U.S. Pat. No. 7,498,298; Doronina et al. (2006)Bioconjugate Chem. 17:114-124; and Doronina et al. (2003) Nat. Biotech.21:778-784 and then conjugated to an antibody of interest.

(3) Calicheamicin

In some embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics, and analogues thereof, are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations (Hinman etal., (1993) Cancer Research 53:3336-3342; Lode et al., (1998) CancerResearch 58:2925-2928). Calicheamicin has intracellular sites of actionbut, in certain instances, does not readily cross the plasma membrane.Therefore, cellular uptake of these agents through antibody-mediatedinternalization may, in some embodiments, greatly enhances theircytotoxic effects. Nonlimiting exemplary methods of preparingantibody-drug conjugates with a calicheamicin drug moiety are described,for example, in U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; and5,767,285.

In some embodiments, the calicheamicin drug moiety conjugated to theantibody is a compound having the formula:

wherein X is Br or I; L is a linker; R is hydrogen, C₁₋₆alkyl, or —C(═O)C₁₋₆alkyl; and R^(a) is hydrogen or C₁₋₆alkyl.

In some embodiments, X is Br, R^(a) is hydrogen and R is isopropyl.

In other embodiments, X is Br, R^(a) is hydrogen and R is ethyl.

In other embodiments, X is I, R^(a) is hydrogen and R is isopropyl.

In other embodiments, X is I, R^(a) is hydrogen and R is ethyl.

In some embodiments, X is Br, R^(a) is hydrogen and R —C(═O)CH₃.

In other embodiments, X is I, R^(a) is hydrogen and R is —C(═O)CH₃.

In other embodiments, X is I, R^(a) is ethyl and R is —C(═O)CH₃.

In other embodiments, X is Br, R^(a) is ethyl and R is —C(═O)CH₃.

(4) Pyrrolobenzodiazepines

In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). Insome embodiments, PDB dimers recognize and bind to specific DNAsequences. The natural product anthramycin, a PBD, was first reported in1965 (Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5793-5795;Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5791-5793). Since then,a number of PBDs, both naturally-occurring and analogues, have beenreported (Thurston, et al., (1994) Chem. Rev. 1994, 433-465 includingdimers of the tricyclic PBD scaffold (U.S. Pat. Nos. 6,884,799;7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; 7,557,099).Without intending to be bound by any particular theory, it is believedthat the dimer structure imparts the appropriate three-dimensional shapefor isohelicity with the minor groove of B-form DNA, leading to a snugfit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem.Res., 19:230-237). Dimeric PBD compounds bearing C2 aryl substituentshave been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem.53(7):2927-2941; Howard et al (2009) Bioorganic and Med. Chem. Letters19(22):6463-6466).

In some embodiments, PBD compounds can be employed as prodrugs byprotecting them at the N10 position with a nitrogen protecting groupwhich is removable in vivo (WO 00/12507; WO 2005/023814).

PBD dimers have been conjugated to antibodies and the resulting ADCshown to have anticancer properties (US 2010/0203007). Nonlimitingexemplary linkage sites on the PBD dimer include the five-memberedpyrrolo ring, the tether between the PBD units, and the N10-C11 iminegroup (WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431;US 2011/0256157; WO 2011/130598).

Nonlimiting exemplary PBD dimer components of ADCs are of Formula A:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the dotted lines indicate the optional presence of a double bond betweenC1 and C2 or C2 and C3;

R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR, ═CH—R^(D),═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally further selected fromhalo or dihalo, wherein R^(D) is independently selected from R, CO₂R,COR, CHO, CO₂H, and halo;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo;

Q is independently selected from O, S and NH;

R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal cation;

R and R′ are each independently selected from optionally substitutedC₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀ heterocycle, and C₅₋₂₀aryl groups, and optionally in relation to the group NRR′, R and R′together with the nitrogen atom to which they are attached form anoptionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively;

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g.benzene or pyridine, which rings are optionally substituted; and

X and X′ are independently selected from O, S and N(H).

In some embodiments, R and R′ are each independently selected fromoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocycle, and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring.

In some embodiments, R⁹ and R¹⁹ are H.

In some embodiments, R⁶ and R¹⁶ are H.

In some embodiments, R⁷ are R¹⁷ are both OR^(7A), where R^(7A) isoptionally substituted C₁₋₄ alkyl.

In some embodiments, R^(7A) is Me. In some embodiments, R^(7A) is isCh₂Ph, where Ph is a phenyl group.

In some embodiments, X is O.

In some embodiments, R¹¹ is H.

In some embodiments, there is a double bond between C2 and C3 in eachmonomer unit.

In some embodiments, R² and R¹² are independently selected from H and R.In some embodiments, R² and R¹² are independently R. In someembodiments, R² and R¹² are independently optionally substituted C₅₋₂₀aryl or C₅₋₇ aryl or C₈₋₁₀ aryl. In some embodiments, R² and R¹² areindependently optionally substituted phenyl, thienyl, napthyl, pyridyl,quinolinyl, or isoquinolinyl. In some embodiments, R² and R¹² areindependently selected from ═O, ═CH₂, ═CH—R^(D), and ═C(R^(D))₂. In someembodiments, R² and R¹² are each ═CH₂. In some embodiments, R² and R¹²are each H. In some embodiments, R² and R¹² are each ═O. In someembodiments, R² and R¹² are each ═CF₂. In some embodiments, R² and/orR¹² are independently ═C(R^(D))₂. In some embodiments, R² and/or R¹² areindependently ═CH—R^(D).

In some embodiments, when R² and/or R¹² is ═CH—R^(D), each group mayindependently have either configuration shown below:

In some embodiments, a ═CH—R^(D) is in configuration (I).

In some embodiments, R″ is a C₃ alkylene group or a C₅ alkylene group.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(I):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(II):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A III):

wherein R^(E) and R^(E″) are each independently selected from H orR^(D), wherein R^(D) is defined as above; and wherein n is 0 or 1.

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, R^(E) and/or R^(E″) is H. In some embodiments, R^(E) andR^(E″) are H. In some embodiments, R^(E) and/or R^(E″) is R^(D), whereinR^(D) is optionally substituted C₁₋₁₂ alkyl. In some embodiments, R^(E)and/or R^(E″) is R^(D), wherein R^(D) is methyl.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(IV):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; andwherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(V):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; and

wherein n is 0 or 1.

In some embodiments, Ar¹ and Ar² are each independently selected fromoptionally substituted phenyl, furanyl, thiophenyl and pyridyl. In someembodiments, Ar¹ and Ar² are each independently optionally substitutedphenyl. In some embodiments, Ar¹ and Ar² are each independentlyoptionally substituted thien-2-yl or thien-3-yl. In some embodiments,Ar¹ and Ar² are each independently optionally substituted quinolinyl orisoquinolinyl. The quinolinyl or isoquinolinyl group may be bound to thePBD core through any available ring position. For example, thequinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl,quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. In someembodiments, the quinolinyl is selected from quinolin-3-yl andquinolin-6-yl. The isoquinolinyl may be isoquinolin-1-yl,isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl,isoquinolin-7-yl and isoquinolin-8-yl. In some embodiments, theisoquinolinyl is selected from isoquinolin-3-yl and isoquinolin-6-yl.

Further nonlimiting exemplary PBD dimer components of ADCs are ofFormula B:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the wavy line connected to the OH indicates the S or R configuration;

R^(V1) and R^(V2) are independently selected from H, methyl, ethyl andphenyl (which phenyl may be optionally substituted with fluoro,particularly in the 4 position) and C₅₋₆ heterocyclyl; wherein R^(V1)and R^(V2) may be the same or different; and

n is 0 or 1.

In some embodiments, R^(V1) and R^(V2) are independently selected fromH, phenyl, and 4-fluorophenyl.

In some embodiments, a linker may be attached at one of various sites ofthe PBD dimer drug moiety, including the N10 imine of the B ring, theC-2 endo/exo position of the C ring, or the tether unit linking the Arings (see structures C(I) and C(II) below).

Nonlimiting exemplary PBD dimer components of ADCs include Formulas C(I)and C(II):

Formulas C(I) and C(II) are shown in their N10-C11 imine form. ExemplaryPBD drug moieties also include the carbinolamine and protectedcarbinolamine forms as well, as shown in the table below:

wherein:

X is CH₂ (n=1 to 5), N, or O;

Z and Z′ are independently selected from OR and NR₂, where R is aprimary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₁, R′₁, R₂ and R′₂ are each independently selected from H, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀ aryl (including substituted aryls),C₅₋₂₀ heteroaryl groups, —NH₂, —NHMe, —OH, and —SH, where, in someembodiments, alkyl, alkenyl and alkynyl chains comprise up to 5 carbonatoms;

R₃ and R′₃ are independently selected from H, OR, NHR, and NR₂, where Ris a primary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₄ and R′₄ are independently selected from H, Me, and OMe;

R₅ is selected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀aryl (including aryls substituted by halo, nitro, cyano, alkoxy, alkyl,heterocyclyl) and C₅₋₂₀ heteroaryl groups, where, in some embodiments,alkyl, alkenyl and alkynyl chains comprise up to 5 carbon atoms;

R₁₁ is H, C₁-C₈ alkyl, or a protecting group (such as acetyl,trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ),9-fluorenylmethylenoxycarbonyl (Fmoc), or a moiety comprising aself-immolating unit such as valine-citrulline-PAB);

R₁₂ is is H, C₁-C₈ alkyl, or a protecting group;

wherein a hydrogen of one of R₁, R′₁, R₂, R′₂, R₅, or R₁₂ or a hydrogenof the —OCH₂CH₂(X)_(n)CH₂CH₂O— spacer between the A rings is replacedwith a bond connected to the linker of the ADC.

Exemplary PDB dimer portions of ADC include, but are not limited to (thewavy line indicates the site of covalent attachment to the linker):

Nonlimiting exemplary embodiments of ADCs comprising PBD dimers have thefollowing structures:

wherein:

n is 0 to 12. In some embodiments, n is 2 to 10. In some embodiments, nis 4 to 8. In some embodiments, n is selected from 4, 5, 6, 7, and 8.

In some embodiments, an ADC comprising a PBD dimer described herein maybe made by conjugating a linker-drug intermediate including a pyridineleaving group via a sulfur atom with a cysteine thiol of an antibody toform a disulfide linkage. Further, in some embodiments, an ADCcomprising a PBD dimer described herein may be made by conjugating alinker-drug intermediate including a thiopyridyl leaving group, whereinthe pyridine ring is substituted with one or more nitro groups. In someembodiments, the pyridyl ring is monosubstituted with —NO₂. In someembodiments, the —NO₂ monosubstitution is para relative to thedisulfide. In some embodiments, the PBD dimer is connected through theN10 position. For example, non-limiting exemplary ADC comprising a PBDdimer may be made by conjugating a monomethylethyl pyridyl disulfide,N10-linked PBD linker intermediate (shown below) to an antibody:

The linkers of PBD dimer-val-cit-PAB-Ab and the PBD dimer-Phe-Lys-PAB-Abare protease cleavable, while the linker of PBD dimer-maleimide-acetalis acid-labile.

PBD dimers and ADCs comprising PBD dimers may be prepared according tomethods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598; WO2013/055987.

(5) Anthracyclines

In some embodiments, an ADC comprising anthracycline. Anthracyclines areantibiotic compounds that exhibit cytotoxic activity. While notintending to be bound by any particular theory, studies have indicatedthat anthracyclines may operate to kill cells by a number of differentmechanisms, including: 1) intercalation of the drug molecules into theDNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis;2) production by the drug of free radicals which then react withcellular macromolecules to cause damage to the cells, and/or 3)interactions of the drug molecules with the cell membrane (see, e.g., C.Peterson et al., “Transport And Storage Of Anthracycline In ExperimentalSystems And Human Leukemia” in Anthracycline Antibiotics In CancerTherapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102). Becauseof their cytotoxic potential anthracyclines have been used in thetreatment of numerous cancers such as leukemia, breast carcinoma, lungcarcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik,in Anthracycline: Current Status And New Developments p 11).

Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin,idarubicin, daunomycin, nemorubicin, and derivatives thereof.Immunoconjugates and prodrugs of daunorubicin and doxorubicin have beenprepared and studied (Kratz et al (2006) Current Med. Chem. 13:477-523;Jeffrey et al (2006) Bioorganic & Med. Chem. Letters 16:358-362; Torgovet al (2005) Bioconj. Chem. 16:717-721; Nagy et al (2000) Proc. Natl.Acad. Sci. USA 97:829-834; Dubowchik et al (2002) Bioorg. & Med. Chem.Letters 12:1529-1532; King et al (2002) J. Med. Chem. 45:4336-4343; EP0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugateBR96-doxorubicin reacts specifically with the tumor-associated antigenLewis-Y and has been evaluated in phase I and II studies (Saleh et al(2000) J. Clin. Oncology 18:2282-2292; Ajani et al (2000) Cancer Jour.6:78-81; Tolcher et al (1999) J. Clin. Oncology 17:478-484).

PNU-159682 is a potent metabolite (or derivative) of nemorubicin(Quintieri, et al. (2005) Clinical Cancer Research 11(4):1608-1617).Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al (1990) Cancer Treat. Rev.17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703;), including phaseII/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedingsof the American Society for Clinical Oncology 22, Abs 1448; Quintieri(2003) Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology24:14116).

A nonlimiting exemplary ADC comprising nemorubicin or nemorubicinderivatives is shown in Formula Ia:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L₁ and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

A further nonlimiting exemplary ADC comprising nemorubicin ornemorubicin derivatives is shown in Formula Ib:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L₂ and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

In some embodiments, the nemorubicin component of anemorubicin-containing ADC is PNU-159682. In some such embodiments, thedrug portion of the ADC may have one of the following structures:

wherein the wavy line indicates the attachment to the linker (L).

Anthracyclines, including PNU-159682, may be conjugated to antibodiesthrough several linkage sites and a variety of linkers (US 2011/0076287;WO2009/099741; US 2010/0034837; WO 2010/009124), including the linkersdescribed herein.

Exemplary ADCs comprising a nemorubicin and linker include, but are notlimited to:

wherein:

R₁ and R₂ are independently selected from H and C₁-C₆ alkyl; and

The linker of PNU-159682 maleimide acetal-Ab is acid-labile, while thelinkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab,and PNU-159682-val-cit-PAB-spacer(R₁R₂)-Ab are protease cleavable.

(6) 1-(Chloromethyl)-2, 3-dihydro-1H-benzo[e]indole (CBI) Dimer DrugMoieties

In some embodiments, an ADC comprises1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI). The5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (amino CBI) classof DNA minor groove alkylators are potent cytotoxins (Atwell, et al(1999) J. Med. Chem., 42:3400), and have been utilized as effector unitsin a number of classes of prodrugs designed for cancer therapy. Thesehave included antibody conjugates, (Jeffrey, et al. (2005) J. Med.Chem., 48:1344), prodrugs for gene therapy based on nitrobenzylcarbamates (Hay, et al (2003) J. Med. Chem. 46:2456) and thecorresponding nitro-CBI derivatives as hypoxia-activated prodrugs(Tercel, et al (2011) Angew. Chem., Int. Ed., 50:2606-2609). The CBI andpyrrolo[2,1-c][1,4]benzodiazepine (PBD) pharmacophores have been linkedtogether by an alkyl chain (Tercel et al (2003) J. Med. Chem46:2132-2151).

In some embodiments, an ADC comprises a1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer (WO2015/023355). In some such embodiments, the dimer is a heterodimerwherein one half of the dimer is a CBI moiety and the other half of thedimer is a PBD moiety.

In some embodiments, a CBI dimer comprises the formula:

where

-   R¹ is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to a    linker (L);-   R² is selected from H, P(O)₃H₂, C(O)NR^(a)R^(b), or a bond to a    linker (L);-   R^(a) and R^(b) are independently selected from H and C₁-C₆ alkyl    optionally substituted with one or more F, or R^(a) and R^(b) form a    five or six membered heterocyclyl group;-   T is a tether group selected from C₃-C₁₂ alkylene, Y, (C₁-C₆    alkylene)-Y—(C₁-C₆ alkylene), (C₁-C₆ alkylene)-Y—(C₁-C₆    alkylene)-Y—(C₁-C₆ alkylene), (C₂-C₆ alkenylene)-Y—(C₂-C₆    alkenylene), and (C₂-C₆ alkynylene)-Y—(C₂-C₆ alkynylene);-   where Y is independently selected from O, S, NR¹, aryl, and    heteroaryl;-   where alkylene, alkenylene, aryl, and heteroaryl are independently    and optionally substituted with F, OH, O(C₁-C₆ alkyl), NH₂, NHCH₃,    N(CH₃)₂, OP(O)₃H₂, and C₁-C₆ alkyl, where alkyl is optionally    substituted with one or more F;-   or alkylene, alkenylene, aryl, and heteroaryl are independently and    optionally substituted with a bond to L;-   D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

-   X¹ and X² are independently selected from O and NR³, where R³ is    selected from H and C₁-C₆ alkyl optionally substituted with one or    more F;-   R⁴ is H, CO₂R, or a bond to a linker (L), where R is C₁-C₆ alkyl or    benzyl; and-   R⁵ is H or C₁-C₆ alkyl.

(6) Amatoxin

In some embodiments, the immunoconjugate comprises an antibodyconjugated to one or more amatoxin molecules. Amatoxins are cyclicpeptides composed of 8 amino acids. They can be isolated from Amanitaphalloides mushrooms or prepared synthetically. Amatoxins specificallyinhibit the DNA-dependent RNA polymerase II of mammalian cells, andthereby also the transcription and protein biosynthesis of the affectedcells. Inhibition of transcription in a cell causes stop of growth andproliferation. See e.g., Moldenhauer et al. JNCI 104:1-13 (2012),WO2010115629, WO2012041504, WO2012119787, WO2014043403, WO2014135282,and WO2012119787, which are hereby incorporated by reference in itsentirety. In some embodiments, the one or more amatoxin molecules areone or more α-amanitin molecules.

(7) Other Drug Moities

Drug moieties also include geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791); and enzymatically active toxins and fragments thereof,including, but not limited to, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, e.g., WO 93/21232.

Drug moieties also include compounds with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. In some embodiments, when an immunoconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹ or I¹²³, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, a peptide may be biosynthesized or chemicallysynthesized using suitable amino acid precursors comprising, forexample, one or more fluorine-19 atoms in place of one or morehydrogens. In some embodiments, labels such as Tc⁹⁹, I¹²³, Re¹⁸⁶, Re¹⁸⁸and In¹¹¹ can be attached via a cysteine residue in the antibody. Insome embodiments, yttrium-90 can be attached via a lysine residue of theantibody. In some embodiments, the IODOGEN method (Fraker et al (1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRCPress 1989) describes certain other methods.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme. In some such embodiments, aprodrug-activating enzyme converts a prodrug (e.g., a peptidylchemotherapeutic agent, see WO 81/01145) to an active drug, such as ananticancer drug. Such immunoconjugates are useful, in some embodiments,in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anticancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysin, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibodies by recombinant DNA techniques well known in the art. See,e.g., Neuberger et al., Nature 312:604-608 (1984).

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADC compounds with a distribution ofone or more drug moieties attached to an antibody. The average number ofdrugs per antibody may be calculated from the mixture by a dual ELISAantibody assay, which is specific for antibody and specific for thedrug. Individual ADC molecules may be identified in the mixture by massspectroscopy and separated by HPLC, e.g. hydrophobic interactionchromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design &Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on thepharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous ADC with a single loading value may beisolated from the conjugation mixture by electrophoresis orchromatography.

Antibody drug conjugates 51-58 of Table A may be prepared by coupling adrug moiety with a linker reagent, and according to the procedures of WO2013/055987; WO 2015/023355; WO 2010/009124; WO 2015/095227, andconjugated with any of the anti-CLL1 antibodies, including cysteineengineered antibodies, described herein. Specific antibody-drugconjugates are recited in Table B.

TABLE A Antibody Drug Conjugates 51-58 ADC No. Structure 51

52

53

54

55

56

57

58

Additional exemplary antibody drug conjugates include:

It is noted that for simplicity the structures above and those of ADCs51 to 58 only show one linker-drug group attached to an antibody. Asmentioned above, more than one linker-drug group can be attached to anantibody.

TABLE B Specific Antibody-drug conjugates (ADC) linker-drug LD No. ADCNo. ADC formula (Table 1) DAR* ADC-101 Thio Ch Anti-CLL-1 21C9 HCA118C-(LD-55) 55 2.0 ADC-102 Thio Ch Anti-CLL-1 3H10 HC A118C-(LD-55) 552.0 ADC-103 Thio Ch Anti-CLL-1 28H12 HC A118C-(LD-55) 55 1.9 ADC-104Thio Ch Anti-CLL-1 20B1 HC A118C-(LD-55) 55 1.8 ADC-105 Thio ChAnti-CLL-1 6E7 HC A118C-(LD-55) 55 1.9 ADC-106 Thio Hu anti-CLL-16E7.H1eL4 HC A118C-(LD-54) 54 1.95 ADC-107 Thio Hu anti-CLL-1 21C9.H3L2HC A118C-(LD-54) 54 1.96 ADC-108 Thio Hu anti-CLL-1 21C9.H3L2 LCK149C-(LD-54) 51 1.9 ADC-109 Thio Hu anti-CLL-1 6E7.H1eL4 LCK149C-(LD-51) 51 1.91 ADC-110 Thio Hu anti-CLL-1 6E7.N54A LCK149C-(LD-51) 51 2.0 ADC-111 Thio Hu anti-CLL-1 6E7.H1eL4.N54A LCK149C-(LD-53) 53 2.0 ADC-112 Thio Hu anti-CLL-1 6E7.H1eL4.N54A LCK149C-(LD-52) 52 1.9 ADC-113 Thio Hu anti-CLL-1 6E7.N54A LCK149C-(LD-51) 51 1.9 ADC-114 Thio Hu anti-CLL-1 6E7.N54A LCK149C-(LD-56) 56 2.0 ADC-115 Thio Hu anti-CLL-1 6E7.N54A LCK149C-(LD-57) 57 1.7 ADC-116 Thio Hu anti-CLL-1 6E7.N54A LCK149C-(LD-58) 58 1.9 DAR = drug/antibody ratio average A118C (EUnumbering) = A121C (Sequential numbering) = A114C (Kabat numbering)Wild-type (“WT”), cysteine engineered mutant antibody (“thio”), lightchain (“LC”), heavy chain (“HC”), 6-maleimidocaproyl (“MC”),maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”),alanine-phenylalanine (“ala-phe”), p-aminobenzyl (“PAB”), andp-aminobenzyloxycarbonyl (“PABC”)

d) Certain Methods of Preparing Immunoconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in U.S. Pat. No. 7,498,298, whichis expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADC are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety, are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-CLL-1 antibodies provided hereinis useful for detecting the presence of CLL-1 in a biological sample.The term “detecting” as used herein encompasses quantitative orqualitative detection. A “biological sample” comprises, e.g., a cell ortissue (e.g., biopsy material, including cancerous or potentiallycancerous lymphoid tissue, such as lymphocytes, lymphoblasts, monocytes,myelomonocytes, and mixtures thereof).

In one embodiment, an anti-CLL-1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of CLL-1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-CLL-1 antibody as described herein under conditionspermissive for binding of the anti-CLL-1 antibody to CLL-1, anddetecting whether a complex is formed between the anti-CLL-1 antibodyand CLL-1 in the biological sample. Such method may be an in vitro or invivo method. In one embodiment, an anti-CLL-1 antibody is used to selectsubjects eligible for therapy with an anti-CLL-1 antibody, e.g. whereCLL-1 is a biomarker for selection of patients. In a further embodiment,the biological sample is a cell or tissue.

In a further embodiment, an anti-CLL-1 antibody is used in vivo todetect, e.g., by in vivo imaging, a CLL-1-positive cancer in a subject,e.g., for the purposes of diagnosing, prognosing, or staging cancer,determining the appropriate course of therapy, or monitoring response ofa cancer to therapy. One method known in the art for in vivo detectionis immuno-positron emission tomography (immuno-PET), as described, e.g.,in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel etal., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method isprovided for detecting a CLL-1-positive cancer in a subject, the methodcomprising administering a labeled anti-CLL-1 antibody to a subjecthaving or suspected of having a CLL-1-positive cancer, and detecting thelabeled anti-CLL-1 antibody in the subject, wherein detection of thelabeled anti-CLL-1 antibody indicates a CLL-1-positive cancer in thesubject. In certain of such embodiments, the labeled anti-CLL-1 antibodycomprises an anti-CLL-1 antibody conjugated to a positron emitter, suchas ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In a particularembodiment, the positron emitter is ⁸⁹Zr.

In further embodiments, a method of diagnosis or detection comprisescontacting a first anti-CLL-1 antibody immobilized to a substrate with abiological sample to be tested for the presence of CLL-1, exposing thesubstrate to a second anti-CLL-1 antibody, and detecting whether thesecond anti-CLL-1 is bound to a complex between the first anti-CLL-1antibody and CLL-1 in the biological sample. A substrate may be anysupportive medium, e.g., glass, metal, ceramic, polymeric beads, slides,chips, and other substrates. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, the first or secondanti-CLL-1 antibody is any of the antibodies described herein.

Exemplary disorders that may be diagnosed or detected according to anyof the above embodiments include CLL-1-positive cancers, such asCLL-1-positive AML, CLL-1-positive CML, CLL-1-positive MDS,CLL-1-positive chronic myelomonocytic leukemia, CLL-1-positive APL,CLL-1-positive chronic myeloproliferative disorder, CLL-1-positivethrombocytic leukemia, CLL-1-positive pre-B-ALL, CLL-1-positivepreT-ALL, CLL-1-positive multiple myeloma, CLL-1-positive mast celldisease, CLL-1-positive mast cell leukemia, CLL-1-positive mast cellsarcoma, CLL-1-positive myeloid sarcomas, CLL-1-positive lymphoidleukemia, and CLL-1-positive undifferentiated leukemia. In someembodiments, a CLL-1-positive cancer is a cancer that receives ananti-CLL-1 immunohistochemistry (IHC) or in situ hybridization (ISH)score greater than “0,” which corresponds to very weak or no stainingin >90% of tumor cells, under the conditions described herein in ExampleB. In another embodiment, a CLL-1-positive cancer expresses CLL-1 at a1+, 2+ or 3+ level, as defined under the conditions described herein inExample B. In some embodiments, a CLL-1-positive cancer is a cancer thatexpresses CLL-1 according to a reverse-transcriptase PCR (RT-PCR) assaythat detects CLL-1 mRNA. In some embodiments, the RT-PCR is quantitativeRT-PCR.

In certain embodiments, labeled anti-CLL-1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like. In anotherembodiment, a label is a positron emitter. Positron emitters include butare not limited to ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In aparticular embodiment, a positron emitter is ⁸⁹Zr.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-CLL-1 antibody or immunoconjugateas described herein are prepared by mixing such antibody orimmunoconjugate having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-CLL-1 antibodies or immunoconjugates provided herein maybe used in methods, e.g., therapeutic methods.

In one aspect, an anti-CLL-1 antibody or immunoconjugate provided hereinis used in a method of inhibiting proliferation of a CLL-1-positivecell, the method comprising exposing the cell to the anti-CLL-1 antibodyor immunoconjugate under conditions permissive for binding of theanti-CLL-1 antibody or immunoconjugate to CLL-1 on the surface of thecell, thereby inhibiting the proliferation of the cell. In certainembodiments, the method is an in vitro or an in vivo method. In furtherembodiments, the cell is a lymphocyte, lymphoblast, monocyte, ormyelomonocyte cell. In further embodiments, the cell is a monocyte,granulocyte, and/or progenitors of the monocyte/granulocyte lineage. Insome embodiments, the cell is positive for the presence of FLT3 internaltandem repeats. In some embodiments, the cell is positive for thepresence of a MLL-AF9 fusion gene (e.g., MLL-AF9 translocation). In someembodiments, the cell is positive for the presence of a chromosome 11q23translocation. In some embodiments, the cell is positive for positivefor the presence of a translocation t(9;11)(p22;q23).

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al. (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

In another aspect, an anti-CLL-1 antibody or immunoconjugate for use asa medicament is provided. In further aspects, an anti-CLL-1 antibody orimmunoconjugate for use in a method of treatment is provided. In certainembodiments, an anti-CLL-1 antibody or immunoconjugate for use intreating CLL-1-positive cancer is provided. In certain embodiments, theinvention provides an anti-CLL-1 antibody or immunoconjugate for use ina method of treating an individual having a CLL-1-positive cancer, themethod comprising administering to the individual an effective amount ofthe anti-CLL-1 antibody or immunoconjugate. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow.

In a further aspect, the invention provides for the use of an anti-CLL-1antibody or immunoconjugate in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofCLL-1-positive cancer. In a further embodiment, the medicament is foruse in a method of treating CLL-1-positive cancer, the method comprisingadministering to an individual having CLL-1-positive cancer an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below.

In a further aspect, the invention provides a method for treatingCLL-1-positive cancer. In one embodiment, the method comprisesadministering to an individual having such CLL-1-positive cancer aneffective amount of an anti-CLL-1 antibody or immunoconjugate. In someembodiments, the cancer is AML. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, as described below.

A CLL-1-positive cancer according to any of the above embodiments maybe, e.g., CLL-1-positive AML, CLL-1-positive CML, CLL-1-positivemyelodysplastic syndrome (MDS), CLL-1-positive chronic myelomonocyticleukemia (CML), CLL-1-positive APL, CLL-1-positive chronicmyeloproliferative disorder, CLL-1-positive thrombocytic leukemia,CLL-1-positive pre-B-ALL, CLL-1-positive preT-ALL, CLL-1-positivemultiple myeloma, CLL-1-positive mast cell disease, CLL-1-positive mastcell leukemia, CLL-1-positive mast cell sarcoma, CLL-1-positive myeloidsarcomas, CLL-1-positive lymphoid leukemia, and CLL-1-positiveundifferentiated leukemia. In some embodiments, a CLL-1-positive canceris a cancer that receives an anti-CLL-1 immunohistochemistry (IHC) or insitu hybridization (ISH) score greater than “0,” which corresponds tovery weak or no staining in >90% of tumor cells, under the conditionsdescribed herein in Example B. In another embodiment, a CLL-1-positivecancer expresses CLL-1 at a 1+, 2+ or 3+ level, as defined under theconditions described herein in Example B. In some embodiments, aCLL-1-positive cancer is a cancer that expresses CLL-1 according to areverse-transcriptase PCR (RT-PCR) assay that detects CLL-1 mRNA. Insome embodiments, the RT-PCR is quantitative RT-PCR.

In some embodiments, cell proliferative disorder according to any of theabove embodiments may be, e.g., AML, CML, and/or MDS. In someembodiments, CLL-1-positive cell proliferative disorder is aCLL-1-positive AML, CLL-1-positive CML, CLL-1-positive MDS. In someembodiments, the AML is one or more of AML subtype 1, AML subtype 2, AMLsubtype 3, AML subtype 4, AML subtype 5, AML subtype 6, and AML subtype7. In some embodiments, the AML is AML subtype 3 (acute promyelocyticleukemia, APML). In some embodiments, the AML is one or more of AMLsubtype 1, AML subtype 2, AML subtype 4, AML subtype 5, AML subtype 6,and AML subtype 7, and not AML subtype 3.

In some embodiments, the cell proliferative disorder (e.g.,CLL-1-positive cancer and/or AML) is positive for the presence of amutation in FLT3, nucleophosmin (NPM1), CCAAT/enhancer binding proteinalpha (C/EBPα) (CEBPA), and/or c-KIT. In some embodiments, the cellproliferative disorder (e.g., CLL-1-positive cancer and/or AML) ispositive for the presence of FLT3 internal tandem repeats. In someembodiments, the cell proliferative disorder (e.g., CLL-1-positivecancer and/or AML) is positive for the presence of FLT3 tyrosine kinasedomain point mutations. In some embodiments, the cell proliferativedisorder (e.g., CLL-1-positive cancer and/or AML) is positive for thepresence of a mutation in isocitrate dehydrogenase 1 and/or 2 (IDH1and/or IDH2). In some embodiments, the cell proliferative disorder(e.g., CLL-1-positive cancer and/or AML) is positive for the presence ofa mutation in DNA methyltransferase 3A (DNMT3A). In some embodiments,the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML)is NK-AML positive for the presence of (a) a mutation in NPM1 and FLT3,(b) wild-type NPM1 and mutated FLT3, and/or (c) wild-type NPM1 and FLT3.

In some embodiments, the cell proliferative disorder (e.g.,CLL-1-positive cancer and/or AML) is positive cytogenetic abnormalitysuch as one or more of t(15;17), t(8;21), inv(16), t(16;16),t(9;11)(p22;q23), t(6;9)(p23;q34), inv(3)(q21 q26.2),inv(3;3)(q21;q26.2), t(1;22)(p13;q13), t(8;21)(q22;q22),inv(16)(p13;1q22), t(16;16)(p13.1;q22), and/or t(15;17)(q22;q12). Insome embodiments, the cell proliferative disorder (e.g., CLL-1-positivecancer and/or AML) is positive for the presence of a MLL-AF9 fusion gene(e.g., MLL-AF9 translocation). In some embodiments, the cellproliferative disorder (e.g., CLL-1-positive cancer and/or AML) ispositive for the presence of a chromosome 11q23 translocation. In someembodiments, the cell proliferative disorder (e.g., CLL-1-positivecancer) is a cell proliferative disorder (e.g., CLL-1-positive cancerand/or AML) positive for the presence of a translocationt(9;11)(p22;q23).

An “individual” according to any of the above embodiments may be ahuman.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-CLL-1 antibodies or immunoconjugate providedherein, e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-CLL-1antibodies or immunoconjugates provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the anti-CLL-1 antibodies or immunoconjugates providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other agents in a therapy. For instance, anantibody or immunoconjugate of the invention may be co-administered withat least one additional therapeutic agent. In some embodiments, theadditional therapeutic agent is an anthracycline. In some embodiments,the anthracycline is daunorubicin or idarubicin. In some embodiments,the additional therapeutic agent is cytarabine. In some embodiments, theadditional therapeutic agent is cladribine. In some embodiments, theadditional therapeutic agent is fludarabine or topotecan. In someembodiments, the additional therapeutic agent is 5-azacytidine ordecitabine. In some embodiments, the additional therapeutic agent isATRA (all-trans retinoic acid). In some embodiments, the additionaltherapeutic agent is arsenic trioxide. In some embodiments, theadditional therapeutic agent is hydroxyurea. In some embodiments, theadditional therapeutic agent is etoposide. In some embodiments, theadditional therapeutic agent is mitoxantrone. In some embodiments, theadditional therapeutic agent is clofarabine. In some embodiments, theadditional therapeutic agent is hydroxyurea. In some embodiments, theadditional therapeutic agent is FLT3 inhibitor such as quizartinib. Insome embodiments, the additional therapeutic agent is an IDH2 inhibitor.In some embodiments, the additional therapeutic agent is CHK1 inhibitor.In some embodiments, the additional therapeutic agent is a Plk inhibitorsuch as volasertib.

In some embodiments of any of the methods, the additional therapeutic isa BCL2 inhibitor. In some embodiments, the BCL2 inhibitor is venatoclax.

In some embodiments of any of the methods, the additional therapeuticagent is an epigenetic modifier. In some embodiments, the epigeneticmodifier is a histone deacetylase inhibitor. In some embodiments, theepigenetic modifier is DNA methyltransferases I inhibitor. In someembodiments, the epigenetic modifier is a histone methyltransferasesinhibitor. In some embodiments, the epigenetic modifier is a BETinhibitor. In some embodiments, the BET inhibitor selectively targetsthe first bromodomain (BD1). In some embodiments, the BET inhibitorselectively targets the second bromodomain (BD2). In some embodiments,the BET inhibitor is one or more of GSK1210151A, GSK525762, OTX-01,TEN-010, CPI-203, and CPI-0610.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy is stemcell transplant. In some embodiments, the additional therapy may be aseparate administration of one or more of the therapeutic agentsdescribed above.

In some embodiments of any of the methods, the additional therapycomprises cancer immunotherapies. In some embodiments of any of themethods, the cancer immunotherapy comprises a PD-1 axis bindingantagonist. In some embodiments of any of the methods, the cancerimmunotherapy comprises a PD-1 binding antagonist. In some embodimentsof any of the methods, the cancer immunotherapy comprises a PD-L1binding antagonist. In some embodiments of any of the methods, thecancer immunotherapy therapy comprises a PD-L2 binding antagonist. Insome embodiments of any of the methods, the cancer immunotherapycomprises CTLA-4 inhibition. In some embodiments of any of the methods,the cancer immunotherapy comprises immune agonists.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of antibody orimmunoconjugate, the severity and course of the disease, whether theantibody or immunoconjugate is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody or immunoconjugate, and the discretion ofthe attending physician. The antibody or immunoconjugate is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or immunoconjugate can be aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody orimmunoconjugate would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using both an immunoconjugate of theinvention and an anti-CLL-1 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thedisorder and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or immunoconjugate of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody or immunoconjugate of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution ordextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1

A. Monoclonal Antibody Generation

Monoclonal antibodies against human (hu) and cynomolgus (cyno) CLL-1were generated using the following procedures by immunizing animals withrecombinant hu and cyno CLL-1 extracellular domain (ECD, amino acids of65-265 huCLL-1 and 65-265 cynoCLL-1) fused to a N-terminal Flag(DYKDDDDK) expressed in a mammalian expression system. The huCLL1 ECDprotein (amino acids 65-265) comprised a SNP, AAA(Lys, K) 244->CAA (GLN,Q), which has a minor allele frequency (MAF) of 29%.

Positive clones were expanded and re-screened for binding to huCLL-1 andcynoCLL-1 by ELISA and FACS. Five clones were identified: m3H10, m6E7,m20B1, m21C9, and m28H12 that reacted strongly by fluorescent activatedcell sorting (FACS) with stable cell lines expressing recombinant hu andcyno CLL-1, and with tumor-derived CLL-1 expressed on Acute MyeloidLeukemia tumor cell lines. Alignment of the amino acid sequences of themurine heavy and light variable domains are shown in FIGS. 1A and B.m3H10 and m21C9 share the same heavy and light chain CDRs, only theamino acid sequences of m21C9 heavy and light chain variable region isshown in FIG. 1.

B. Species Cross-Reactivity and Binding Affinity

Monoclonal antibodies were tested to determine if they cross-react withcynoCLL-1 extracellular domain (ECD) (which is 85.07% identical and87.35% similar to the huCLL-1 protein ECD). The chimeric anti-CLL-1human IgG were captured by mouse anti-human IgG coated on the CM5 sensorchip. For kinetics measurements, three-fold serial dilutions of human orcyno CLL-1 (4.1 nM to 1000 nM) were injected in HBS-EP buffer.Association rates (kon) and dissociation rates (koff) were calculatedusing a simple one-to-one Langmuir binding model. The equilibriumdissociation constant (KD) was calculated as the ratio koff/kon. Table 2below shows that the chimeric version of the five antibodies (m3H10,m6E7, m20B1, m21C9, and m28H12) recognized both recombinant hu andcynoCLL-1 and provides details regarding the kinetics of the interactionwith hu and cyno-CLL-1. Further confirmation of cross-reactivity to cynoCLL-1 was done by FACS analyses of blood from cynomolgus (Mauritianorigin) (data not shown).

TABLE 2 Biacore of Anti-CLL-1 Antibodies Ligand Analyte Ka (M⁻¹s⁻¹) Kd(s⁻¹) KD (M) ch3H10 huCLL-1-Flag 2.7 × 10⁵ 2.4 × 10⁻³ 8.7 nM CynoCLL-1Flag 1.7 × 10⁵ 7.7 × 10⁻⁴ 4.3 nM ch6E7 huCLL-1-Flag 4.6 × 10⁵ 4.4 × 10⁻⁴0.9 nM CynoCLL-1 Flag 4.0 × 10⁵ 4.6 × 10⁻⁴ 1.1 nM ch20B1 huCLL-1-Flag2.2 × 10⁵ 1.0 × 10⁻³ 4.5 nM CynoCLL-1 Flag 1.9 × 10⁵ 1.2 × 10⁻³ 6.1 nMch21C9 huCLL-1-Flag 2.5 × 10⁵ 2.4 × 10⁻³ 9.7 nM CynoCLL-1 Flag 1.6 × 10⁵1.2 × 10⁻³ 7.1 nM ch28H12 huCLL-1-Flag 5.0 × 10⁵ 9.5 × 10⁻³  18 nMCynoCLL-1 Flag 6.7 × 10⁵ 2.3 × 10⁻⁴ 0.3 nM

Scatchard analysis was performed following standard procedures (Holmeset al., Science 256:1205-1210 (1992)) to determine the relative bindingaffinities of the antibodies including ch6E7 and ch21C9.

Anti-CLL-1 antibodies were [I¹²⁵] labeled using the indirect Iodogenmethod. The [I¹²⁵] labeled anti-CLL-1 antibodies were purified from free¹²⁵I—Na by gel filtration using a NAP-5 column (GE Healthcare); thepurified iodinated anti-CLL-1 antibodies had a range of specificactivities of 8-10 μCi/μg. Competition assay mixtures of 50 μL volumecontaining a fixed concentration of [I¹²⁵] labeled antibody anddecreasing concentrations of serially diluted, unlabeled antibody wereplaced into 96-well plates. HEK293AD cells stably expressing recombinanthu or cynoCLL-1 or HL-60 tumor cells expressing endogenous CLL-1 werecultured in growth media at 37° C. in 5% CO₂. Cells were detached fromthe flask using Sigma Cell Dissociation Solution and were washed withbinding buffer, which consisted of Dulbecco's Modified Eagle Medium(DMEM) with 1% bovine serum albumin (BSA), 300 mM human IgG and 0.1%sodium azide. The washed cells were added to the 96 well plates at adensity of 100,000 cells in 0.2 mL of binding buffer. The finalconcentration of the [I¹²⁵] labeled antibody in each well was ˜250 pM.The final concentration of the unlabeled antibody in the competitionassay ranged from 1000 nM through ten 2-fold dilution steps to a 0 nMbuffer-only assay. Competition assays were carried out in triplicate.Competition assays were incubated for 2 hours at room temperature. Afterthe 2-hour incubation, the competition assays were transferred to aMillipore Multiscreen filter plate (Billerica, Mass.) and washed 4 timeswith binding buffer to separate the free from bound [I¹²⁵] labeledantibody. The filters were counted on a Wallac Wizard 1470 gamma counter(PerkinElmer Life and Analytical Sciences Inc.; Wellesley, Mass.). Thebinding data was evaluated using NewLigand software (Genentech), whichuses the fitting algorithm of Munson and Robard to determine the bindingaffinity of the antibody (Munson and Robard 1980).

Table 3 shows the affinity (kD range of 0.45-1.2 nM) to recombinant huand cynoCLL-1 expressed by HEK293AD CLL-1 stable cells and to HL-60cells.

TABLE 3 Antibody Affinity [kD = nM] to CLL-1 (Scatchard Analysis). Cellsch6E7 ch21C9 HL-60 K_(D) (nM) 0.65 0.45 EOL-1 K_(D) (nM) 293AD/huCLL-1K_(D) (nM) 0.80 0.59 293AD/cynoCLL-1 K_(D) (nM) 1.0 1.2

C. Monoclonal Antibody Epitope Grouping

Epitope grouping was also determined using a cell-based competitionbinding FACS assay. HL-60 cells were pre-incubated with or without50-100 fold excess of unlabeled competing antibodies, then stained withdirectly labeled detection antibodies, a reduction of the signal fromdetecting antibody indicating that the unlabeled competing antibodybinds to the same or similar region on CLL-1 as the detectingantibody—this should occur when the same antibody is used as bothdetector and competitor. When there is no blocking of detector signal bya different unlabeled antibody, the unlabeled antibody is binding to adifferent region in CLL-1.

TABLE 4 Anti-CLL-1 Competition Experiments Competing antibodiesDetecting antibodies ch6E7 ch20B1 ch21C9 ch28H12 R&D R&D Systems-PE ✓

✓

✓ (Clone 687317) ch6E7-DyLight650 ✓

n/a

n/a ch28H12-DyLight650 n/a

n/a ✓ n/a ch21C9-DyLight650 ✓

✓

✓ eBioscience HB3-PE

✓ BD Biosciences 50C1-PE

Table 4 shows epitope grouping of the antibodies to CLL-1. ch6E7 andch21C9, but not ch20B1 and ch28H12, bin with R&D Systems-PE (Clone687317). R&D Systems also blocked eBioscience clone HB3, but ch6E7 andch21C9 were unable to block eBioscience clone HB3 binding. ch20B1 andch28H12 failed to compete with any other antibody suggesting eachantibody binds a distinct epitope. All antibodies failed to compete withBD Biosciences clone 50C1 also suggesting that it binds a distinctepitope.

D. Humanization of Anti-CLL-1 Antibodies

Monoclonal antibody 6E7 and 21C9 was humanized as described below.Residue numbers are according to Kabat et al., Sequences of proteins ofimmunological interest, 5th Ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).

Variants constructed during the humanization of 6E7 and 21C9 wereassessed in the form of an IgG. The VL and VH domains from murine 6E7and 21C9 were aligned with the human VL kappa I (VLKI) and human VHsubgroup I (VHI) consensus sequences. Hypervariable regions from themurine antibodies were engineered into VLKI and VHI acceptor frameworks.Specifically, from the mu6E7 and mu21C9 VL domain, positions 24-34 (L1),50-56 (L2) and 89-97 (L3) were grafted into VLKI and from the mu6E7 andmu21C9 VH domain, positions 26-35 (H1), 50-65 (H2) and 93-102 (H3) weregrafted into VHI.

The binding affinity of the antibodies in this section was determined byBIAcore™ T200 Format. Briefly, BIAcore™ research grade CM5 chips wereactivated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS) reagents according to the supplier'sinstructions. Goat anti-human Fc IgGs were coupled to the chips toachieve approximately 10,000 response units (RU) in each flow cell.Unreacted coupling groups were blocked with 1M ethanolamine. Forkinetics measurements, antibodies were captured to achieve approximately300 RU. Three-fold serial dilutions of human CLL-1 was injected in HBS-Pbuffer (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% surfactant P20) at 25° C.with a flow rate of 30 μl/min. Association rates (kon) and dissociationrates (koff) were calculated using a 1:1 Langmuir binding model(BIAcore™ T200 Evaluation Software version 2.0). The equilibriumdissociation constant (Kd) was calculated as the ratio koff/kon.

The binding affinity of the CDR graft humanized 6E7 antibody wascompared to chimeric 6E7. Additional variants of the CDR graft humanized6E7 antibody were made to evaluate the contribution of other vernierpositions to binding to CLL-1. For 6E7, initially four additional lightchains (L1: CDRs graft+(L4, L48, and K49), L2: CDRs graft+L4, L3: CDRsgraft+K49, and L4: CDRs graft+K49) and five additional heavy chains (H1:CDRs graft+(A67, L69, V71, K73), H2: CDRs graft+A67, H3: CDRs graft+L69,H4:CDRs graft+V71, and H5: CDRs graft+K73). K49 on the light chain wasthe key mouse vernier residue, and L69 and V71 on the heavy chain weredetermined to be the key mouse vernier residues based on mutationalanalysis (data not shown). Chimeric 6E7 bound with a KD of 9.59E-10 M,while CDRs graft+K49(LC)+(A67, L69, V71, K73 (HC)), CDRsgraft+K49(LC)+(L69, V71 (HC)) bound with a KD of 1.40E-9M, and 1.37E-9M,respectively.

The binding affinity of the CDR graft humanized 21C9 antibody wascompared to chimeric 21C9 antibody. Additional variants of the CDR grafthumanized 21C9 antibody were made to evaluate the contribution of othervernier positions to binding to CLL-1. For 21C9, initially threeadditional light chains (L1: CDRs graft+(F36 and S43), L2: CDRsgraft+F36, L3: CDRs graft+S43) and five additional heavy chains (HI:CDRs graft+(A67, L69, V71, K73), H2: CDRs graft+A67, H3: CDRs graft+L69,H4:CDRs graft+V71, and H5: CDRs graft+K73). F36 on the light chain wasthe key mouse vernier residue. Chimeric 21C9 bound with a KD of 8.615E-9M, while CDRs graft+(F36 and S43(LC))+L69 (HC) and CDRs graft+F36(LC)+L69 (HC), bound with a KD of 1.053E-8M and 9.785-9M, respectively.L69 on the heavy chain were determined to be the key mouse vernierresidues.

The humanized 6E7.L4H1e and 21C9.L2H3 were tested for their ability tobind human and cyno CLL-1 as described above except that cynoCLL-1replaced huCLL-1 in the cyno binding assay. Binding properties for thehumanized antibodies are shown below in Table 5. The binding affinity ofthe humanized 6E7.L4H1e was 0.34, 0.29, 0.22, and 0.35 Kd (nM) asdetermined by Scatchard using HL-60, EOL-1, 293AD/cynoCLL1, and293AD/huCLL-1 cells, respectively. The binding affinity of humanized21C9.L2H3 was 1.3, 0.74, 2.4, and 3.6 Kd (nM) as determined by Scatchardusing HL-60, EOL-1, 293AD/cynoCLL1, and 293AD/huCLL-1 cells,respectively.

TABLE 5 cynoka cynokd Antibody huKD (M) huka (1/Ms) hukd (1/s) cynoKD(M) (1/Ms) (1/s) 6E7.L4H1e 6.218E−10 8.236E+5 5.121E−4 3.170E−107.391E+5 2.343E−4 21C9.L2H3 1.171E−8 2.244E+5 2.628E−3 9.472E−9 1.683E+51.594E−3

The humanized antibodies 6E7.L4H1e and 21C9.L2H3 were tested underthermal stress (40° C., pH 5.5, 2 weeks) and 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) Analysis. Samples were thermallystressed to mimic stability over the shelf life of the product. Sampleswere buffer exchanged into 20 mM His Acetate, 240 mM sucrose, pH 5.5 anddiluted to a concentration of 1 mg/mL. One mL of sample was stressed at40C for 2 weeks and a second was stored at ˜70C as a control. Bothsamples were then digested using trypsin to create peptides that couldbe analysed using liquid chromatography (LC)-mass spectrometry (MS)analysis. For each peptide in the sample retention time, from the LC aswell as high resolution accurate mass and peptide ion fragmentationinformation (amino acid sequence information) were acquired in the MS.Extracted ion chromatograms (XIC) were taken for peptides of interest(native and modified peptide ions) from the data sets at a window of+−10 ppm and peaks were integrated to determine area. Relativepercentages of modification were calculated for each sample by takingthe (area of the modified peptide) divided by (area of the modifiedpeptide plus the area of the native peptide) multiplied by 100.

Both 6E7.L4H1e and h21C9.L2H3 have N⁵⁴G⁵⁵ in DR-H2, which is susceptibleto deamination (t0=13.2% and t2wk 14.5% for 6E7.L4H1e and r0=11% andt2wk=11.9%). N54 variants of both antibodies were tested to determine ifpotential deamination could be reduced without affecting binding to huand cynoCLL-1. See Table 6.

TABLE 6 huKD cynoka cynokd Antibody (M) huka (1/Ms) hukd (1/s) cynoKD(M) (1/Ms) (1/s) 6E7.L4H1eN54 1.082E−9 9.096E+5 9.837E−4 2.256E−98.044E+5 1.815E−3 6E7.L4H1eA54 3.082E−9 7.103E+5 2.189E−3 3.143E−96.087E+5 1.913E−3 6E7.L4H1eE54 5.090E−9 4.882E+5 2.485E−3 4.256E−96.641E+5 2.827E−3 6E7.L4H1eS54 1.413E−8 5.098E+5 7.205E−3 6.371E−95.133E+5 3.270E−3 6E7.L4H1eD54 1.132E−7 3.044E+5 3.444E−2 4.870E−81.785E+5 8.694E−3 21C9.L2H3N54 1.510E−8 1.889E+5 2.853E−3 9.302E−92.358E+5 2.194E−3 21C9.L2H3S54 2.859E−7 1.416E+5 4.047E−2 5.669E−6 36562.072E−2 21C9.L2H3A54 6.215E−7 1.113E+5 6.915E−2 4.818E−5 445.3 2.146E−221C9.L2H3E54 8.625E−7 1.022E+5 8.816E−2 4.961E−5 747.5 3.709E−221C9.L2H3D54 8.017E−7 2.858E+5 2.291E−2 2.172E−7 4.072E+4 8.846E−3

For the humanized 6E7.L4H1e CDR-H2 N54 antibody variants, A54 hadacceptable binding characteristics which were most similar to N54. Forthe humanized 21C9.L2H3 CDR-H2 N54 antibody variants, all the variantsshowed a drop in affinity to huCLL-1 in off-rate (10-30 fold) andcynoCLL-1 in on rate (60-500 fold). The binding affinity of thehumanized 6E7.L4H1e was 0.67, 0.68, 0.6, and 0.25 Kd (nM) as determinedby Scatchard using 293AD/cynoCLL1, 293AD/huCLL-1, HL-60, and EOL-1cells, respectively. The binding affinity of humanized 6E7.L4H1eN54A was0.9, 0.89, 0.64, and 0.32 Kd (nM) as determined by Scatchard using293AD/cynoCLL1, 293AD/huCLL-1, HL-60, and EOL-1 cells, respectively.Alignment of the heavy and light variable domain amino acid sequences ofhumanized 6E7 and 21C9 antibodies are shown in FIG. 2A-B and FIG. 3A-B,respectively.

E. Epitope Mapping

To determine the binding epitope of the CLL-1, examination of (a) freeantigen CLL-1 and (b) three different antigen-mAb complexes usinghydroxyl radical footprinting (HRF) techniques was performed. Thesamples were exposed to hydroxyl radicals for intervals of 0, 10, 15,and 20 milliseconds (ms) using the X28c Beam line at the BrookhavenNational Laboratory. The labeled samples were subjected todeglycosylation using PNGase F. A pilot experiment was first carried outon the deglycosylated samples for optimizing the experimental protocol.The pilot investigation using MS revealed that the samples containedsignificant amount of polymer contamination, requiring additional cleanup. In order to remove the polymer contamination, the samples wereprecipitated using Trichloroacetic acid in acetone, and subjected toLC-MS analysis. The precipitation step was successful, and the polymercontamination signal in the MS was significantly attenuated. The cleanedsamples were subjected to reduction and alkylation, digestion usingTrypsin, followed by liquid chromatography coupled with high-resolutionmass spectrometry (LC-MS). The MS data was analyzed using ProtMapMS,resulting in dose response plots for each peptide. Results from the freeantigen were compared against each of the complex forms. Ahomology-based model of the antigen was generated using Swiss-Modelsoftware, and the solvent protected regions were mapped for each of thethree complexes.

The overall sequence coverage obtained using Trypsin mapping was 90.05%.The missing regions were comprised primarily of tryptic peptides thatwere shorter than 4 residues in length, which can be inherentlydifficult to detect due to their weak retention properties on the LCcolumn. The HRF process introduces stable side chain oxidativemodifications resulting in specific mass shifts, which were identifiedfrom the tandem mass spectrometry data. The selected ion chromatograms(SIC) were extracted and integrated for the unoxidized and all oxidizedforms of peptide ion (with particular m/z). These peak area values wereused to characterize reaction kinetics in the form of dose response (DR)plots, which measure the loss of intact peptide as a function of thehydroxyl radical exposure. The solvent protected regions in the complexexperience gradual oxidation reaction as opposed to the free antigen.Differences in the rate of oxidation (called rate constant, RC) serve tohighlight the location of the epitope.

ProtMapMS was used to process the MS data, resulting in RC values foreach peptide. Final results are shown in Table 1. Peptide location andthe corresponding sequence are shown in columns 1 and 2. The thirdcolumn shows the protection ratio, PR (=RCAntigen/RCComplex) for complex1 (6E7.L4H1eA54 antibody and CLL-1 antigen). Similarly, fourth and fifthcolumns show the corresponding protection ratios for complex 2(21C9.L2H3 antibody engineered with a light chain comprising a cysteineresidue at K149 according to Kabat numbering (K149C) and CLL-1 antigen)and complex 3 (R&D Systems monoclonal anti-CLL1 antibody (Clone 687317)and CLL-1 antigen). If the PR value for a given peptide for a particularis less than 1, the corresponding region experienced gain in solventaccessibility due to structural changes introduced during complexformation. A PR value close to 1 indicates that the solventaccessibility of the region remains unchanged, while a PR>1 suggeststhat the corresponding region exhibits protection from the solvent as afunction of the complex formation. The PR values for most of thepeptides for each complex are close to 1, indicating minimal change insolvent accessibility for the corresponding regions. Peptide 142-158consistently shows the highest PR value for all three antibodies,implying significant protection for the region. In addition toprotection of the peptide 142-158, the R&D Systems monoclonal anti-CLL1antibody (Clone 687317, unlike 6E7.L4H1eAG and 21C9.L2H3, also showedsignificant protection of the region 103-116 as evidenced by theoverlapping peptides 103-116 and 105-116.

Pep locn SEQ of SEQ ID ID RCA/ RCA/ RCA/ NO: 1 Sequence NO: RC1 RC2 RC3 65-69 DYKDDDDKLEHVTLK 52 1.4 1.0 1.0  68-69 DDDDKLEHVTLK 53 1.1 0.9 0.8 75-87 MNKLQNISEELQR 54 1.4 1.1 0.90  78-87 LQNISEELQR 55 1.3 1.0 0.8 88-102 NISLQLMSNMNISNK 56 1.1 0.5 0.5 103-116 IRNLSTTLQTIATK 50 1.1 0.82.1 105-116 NLSTTLQTIATK 51 1.2 1.0 2.2 105-119* NLSTTLQTIATKLCR 57 NANA NA 120-124* ELYSK 58 NA NA NA 137-141 WIWHK 59 1.0 0.6 1.3 142-158DSCYFLSDDVQTWQESK 49 3.1 2.0 3.1 159-160 MACAAQNASLLK 60 1.0 1.2 0.8171-181 INNKNALEFIK 61 1.7 1.3 1.1 175-181 NALEFIK 62 1.3 1.0 1.3175-185* NALEFIKSQSR 63 NA NA NA 186-201 SYDYWLGLSPEEDSTR 64 1.0 1.0 1.0186-204* SYDYWLGLSPEEDSTRGMR 65 NA NA NA 205-117 VDNIINSSAWVIR 66 1.21.0 1.0 218-232 NAPDLNNMYCGYINR 67 1.2 1.0 0.9 233-243 LYVQYYHCTYK 681.0 1.1 1.0 246-250* MICEK 69 NA NA NA 251-263 MANPVQLGSTYFR 70 0.99 1.11.0

F. Internalization of Anti-CLL-1 Antibody

One desirable attribute of an ADC target is the ability to internalizethe antibody into a degradative compartment in the cell. To determinewhether anti-CLL-1 antibody gets internalized upon binding, HL-60 orHL-60 cells were pre-incubated for 2 hours at 37° C. with 0.3 mg/ml hIgGin RPMI medium to reduce non-specific binding to FcR before seeding incell culture treated 4-well chamber slides (Nalge Nunc International).Antibody directly conjugated to Dylight 488 at a final concentration of1 μg/mL was incubated with hIgG-blocked cells on ice for 30 minutes inthe dark. The cells were immediately imaged to show membrane staining(T0) and followed with time-lapsed photography over a 10 hour period at37° C. with a Leica SP5 confocal microscope. A representative example,ch21C9, is rapidly internalized within 30 minutes by HL-60 cells (datanot shown). Localization of ch21C9 to the lysosome was confirmed usingan in vitro cell-based assay measuring the ability of an antibody drugconjugate to kill target cells.

G. Production of Anti-CLL-1 Antibody Drug Conjugates

For larger scale antibody production, antibodies were produced in CHOcells. Vectors coding for VL and VH were transfected into CHO cells andIgG was purified from cell culture media by protein A affinitychromatography.

Anti-CLL-1 antibody-drug conjugates (ADCs) were produced by conjugatingantibodies ch21C9, ch3H10, ch28H12, ch20B1, ch6E7, 6E7.L4H1e,6E7.L4H1eA54, 21C9.L2H3 via linkers to PBD and PNU derivatives.

As initially isolated, the engineered cysteine residues in antibodiesexist as mixed disulfides with cellular thiols (e.g., glutathione) andare thus unavailable for conjugation. Partial reduction of theseantibodies (e.g., with DTT), purification, and reoxidation withdehydroascorbic acid (DHAA) gives antibodies with free cysteinesulfhydryl groups available for conjugation, as previously described,e.g., in Junutula et al. (2008) Nat. Biotechnol. 26:925-932 and US2011/0301334. Briefly, the antibodies were combined with the drug-linkermoiety to allow conjugation of the drug-linker moiety to the freecysteine residues of the antibody. After several hours, the ADCs werepurified.

H. Efficacy of Anti-CLL-1 Antibody Drug Conjugates in HL-60 and EOL-1Human Acute Myeloid Leukemia Cell Line Xenograft Models

The efficacy of the anti-CLL-1 ADCs was investigated using human AcuteMyeloid Leukemia xenograft models, HL-60 (AML subtype M2) and EOL-1 (AMLsubtype M4a). Both are associated with intermediate to poor prognosis asa result of their genetics and molecular aberrations. Female C.B-17 SCIDmice (Charles River Laboratories; Hollister, Calif.) were eachinoculated subcutaneously in the flank area with five million cells ofHL-60 or EOL-1. When the xenograft tumors reached an average tumorvolume of 100-300 mm³ (referred to as Day 0), animals were randomizedinto groups of 7-10 mice each and received a single intravenousinjection of the ADCs. Approximately 4 hours prior to administration ofADCs, animals were dosed intraperitoneally with excess amount (30 mg/kg)of anti-gD control antibody to block possible nonspecific antibodybinding sites on the tumor cells. Tumors and body weights of mice weremeasured 1-2 times a week throughout the study. Mice were promptlyeuthanized when body weight loss was >20% of their starting weight. Allanimals were euthanized before tumors reached 3000 mm3 or showed signsof impending ulceration. The presence of the antibodies was confirmed byPK bleeds at 1, 7 and 14 days post injection.

As shown in FIG. 4, the ch21C9, ch28H12, ch20B1, ch6E7, and ch3H10conjugated to the PNU drug moiety:

via a free cysteine at heavy chain amino acid 118 according to EUnumbering (A1118C) significantly reduced EOL-1 tumor volume while ch20B1moderately reduced tumor volume and ch28H12 had little effect. Similarresults were seen using HL-60 as shown in FIG. 5.

The humanized antibodies, 6E7.L4H1e and 21C9.L2H3, were conjugated toPBD derivatives (SG34) via different cysteine engineered conjugationsites at various dosages (10 and 20 μg/m²). The antibodies comprised anengineered free cysteine at heavy chain amino acid 118 according to EUnumbering (A1118C) or light chain amino acid 149 according to Kabatnumbering (K149C). The structures of the antibody-drug conjugates isshown below:

As shown in FIG. 6 in the HL-60 xenograft model, the light chain K149Ccysteine engineered immunoconjugate comprising either 6E7L4H1e or21C9.L2H3 showed greater reduction in tumor volume than the heavy chainA1118C cysteine engineered immunoconjugate comprising either 6E7.L4H1eor 21C9.L2H3.

The ability to reduce tumor volume was also compared between 6E7.L4H1eand the engineered variant to remove the potential deamination site6E7.L4H1eA54 to determine if activity as well as binding of the antibodywas retained. As shown in FIG. 7, both 6E7L4H1e and 6E7 significantlyreduced tumor volume in the HL-60 xenograft model.

Example 2 Synthesis of Linker-Drug (LD) Intermediates Used to Make theAntibody Drug Conjugates Exemplified in (Table A)

Linker-drug intermediate of ADC-51:(R)-2-((5-nitropyridin-2-yl)disulfanyl)propyl(11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(MS (ESI): 875 [M+H]⁺) was prepared by the procedures of WO2013/055987.

Sulfuryl chloride (2.35 mL of a 1.0M solution in DCM, 2.35 mmol) wasadded drop-wise to a stirred suspension of 5-nitropyridine-2-thiol (334mg, 2.14 mmol) in dry DCM (7.5 mL) at 0° C. (ice/acetone) under an argonatmosphere. The reaction mixture turned from a yellow suspension to ayellow solution and was allowed to warm to room temperature then stirredfor 2 hours after which time the solvent was removed by evaporation invacuo to provide a yellow solid. The solid was re-dissolved in DCM (15mL) and treated drop-wise with a solution of (R)-2-mercaptopropan-1-ol(213 mg, 2.31 mmol) in dry DCM (7.5 mL) at 0° C. under an argonatmosphere. The reaction mixture was allowed to warm to room temperatureand stirred for 20 hours at which point analysis by LC/MS revealedsubstantial product formation at retention time 1.41 minutes (ES+) m/z247 ([M+H]⁺, ˜100% relative intensity). The precipitate was removed byfiltration and the filtrate evaporated in vacuo to give an orange solidwhich was treated with H₂O (20 mL) and basified with ammonium hydroxidesolution. The mixture was extracted with DCM (3×25 mL) and the combinedextracts washed with H₂O (20 mL), brine (20 mL), dried (MgSO₄), filteredand evaporated in vacuo to give the crude product. Purification by flashchromatography (gradient elution in 1% increments: 100% DCM to 98:2 v/vDCM/MeOH) gave (R)-2-((5-nitropyridin-2-yl)disulfanyl)propan-1-olas anoil (111 mg, 21% yield).

Triphosgene (48 mg, 0.16 mmol) was added to a stirred solution of(R)-2-((5-nitropyridin-2-yl)disulfanyl)propan-1-ol (111 mg, 0.45 mmol)and pyridine (34 μL, 33.5 mg, 0.42 mmol) in dry DCM (5 mL). The reactionmixture was allowed to stir under an argon atmosphere for 45 minutesafter which time the solvent was removed by evaporation in vacuo toprovide (R)-2-((5-nitropyridin-2-yl)disulfanyl)propyl carbonochloridateas a yellow film. The product was carried through to the next stepwithout purification or analysis.

A solution of (R)-2-((5-nitropyridin-2-yl)disulfanyl)propylcarbonochloridate (˜139 mg, 0.45 mmol) in dry DCM (5 mL) was addeddrop-wise to a stirred solution of di-tert-butyl((pentane-1,5-diylbis(oxy))bis(6-((2R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenecyclopentane-1-carbonyl)-4-methoxy-3,1-phenylene))dicarbamate51a, made by the procedures of Example 1 in WO 2013/055987, (430 mg,˜0.45 mmol) and pyridine (40 μL, 39 mg, 0.49 mmol) in dry DCM (12 mL) atroom temperature. The reaction mixture was allowed to stir under anargon atmosphere for 2.5 hours at which point analysis by LC/MS revealedsubstantial product formation at retention time 2.42 minutes (ES+) m/z1226 ([M+H]^(+.), ˜20% relative intensity), 1248 ([M+Na]^(+.), ˜60%relative intensity). The mixture was diluted with DCM (20 mL) andtreated with SiO₂ and the solvent removed by evaporation in vacuo. Theresulting residue was subjected to purification by flash chromatography(gradient elution in 10% increments: 80:20 v/v hexane/EtOAc to 70:30 v/vhexane/EtOAc) to give tert-Butyl(2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-((5-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxy-5-((((R)-2-((5-nitropyridin-2-yl)disulfanyl)propoxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamate51b as a yellow foam (419 mg, 76% yield). (MS (ESI): 1224 [M+H]⁺)

Glacial acetic acid (24 mL) was added to a stirred solution of theTBS-protected 51b (419 mg, 0.34 mmol) in THF (8 mL) and H₂O (8 mL). Thereaction mixture was allowed to stir for 16 hours at which pointanalysis by LC/MS revealed reaction completion with desired productobserved at retention time 1.82 minutes (ES+) m/z 997 ([M+H]^(+.), ˜100%relative intensity), 1019 ([M+Na]^(+.), ˜45% relative intensity). Thereaction mixture was added drop-wise to a chilled (0-5° C.) saturatedsolution of NaHCO₃ (400 mL). The neutral solution was allowed to warm toroom temperature and extracted with EtOAc (4×100 mL), the combinedorganic layers were washed with H₂O (80 mL), brine (100 mL), dried(MgSO₄), filtered and evaporated in vacuo to give the crude product.Purification by flash chromatography (gradient elution in 1% increments:100% DCM to 98:2 v/v DCM/MeOH) gave tert-Butyl(2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-((5-(4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxy-5-((((R)-2-((5-nitropyridin-2-yl)disulfanyl)propoxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamate51c as a yellowish foam (341 mg, 100% yield). (MS (ESI): 995 [M+H]⁺).

A solution of anhydrous DMSO (107 μL, 188 mg, 1.50 mmol) in dry DCM (7.5mL) was added drop-wise to a stirred solution of oxalyl chloride (410 μLof a 2.0M solution in DCM, 0.82 mmol) in dry DCM (7.5 mL) at −45° C.(dry ice/CH₃CN) under an argon atmosphere. After 15 minutes stirring at−45° C., the reaction mixture was treated drop-wise with a solution of51c (341 mg, 0.34 mmol) in dry DCM (15 mL). After stirring at −45° C.for a further 1 hour, the reaction mixture was treated drop-wise with asolution of TEA (476 μL, 342 mg, 3.42 mmol) in dry DCM (7.5 mL). Thereaction mixture was allowed to warm to room temperature over a periodof 1.5 hours and diluted with DCM (50 mL) then washed with saturatedNH₄C1 (15 mL), saturated NaHCO₃ (15 mL), brine (15 mL), dried (MgSO₄),filtered and evaporated in vacuo to give the crude product. Purificationby flash chromatography (gradient elution in 0.4% increments: 100% DCMto 98.4:1.6 v/v DCM/MeOH) gave tert-butyl(11S,11aS)-11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-7-methoxy-2-methylene-10-(((R)-2-((5-nitropyridin-2-yl)disulfanyl)propoxy)carbonyl)-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate51d as a yellowish foam (227 mg, 67% yield): LC/MS retention time 1.69minutes (ES+) m/z 993 ([M+H]^(+.), ˜80% relative intensity), 1015([M+Na]^(+.), ˜20% relative intensity).

A solution of 95:5 v/v TFA/H₂O (4 mL) was added to a crude sample of 51d(216 mg, 0.22 mmol) at 0° C. (ice/acetone). After stirring at 0° C. for30 minutes the reaction was deemed complete as judged by LC/MS, desiredproduct peak at retention time 1.60 minutes (ES+) m/z 875 ([M+H]^(+.),˜100% relative intensity). The reaction mixture was kept cold and addeddrop-wise to a chilled saturated aqueous solution of NaHCO₃ (100 mL).The mixture was extracted with DCM (3×30 mL) and the combined organiclayers washed with brine (50 mL), dried (MgSO₄), filtered and evaporatedin vacuo to provide the crude product. Purification by flashchromatography (gradient elution in 0.4% increments: 100% CHCl₃ to98.4:1.6 v/v CHCl₃/MeOH) gave LD-51 as a yellow foam (127 mg, 66%yield): LC/MS (15-minute run), retention time 6.18 minutes (ES+) m/z 875([M+H]^(+.), ˜100% relative intensity); ¹H NMR (400 MHz, CDCl₃) δ 9.21(s, 1H), 8.30 (d, 1H, J=8.8 Hz), 7.69 (d, 1H, J=4.5 Hz), 7.62 (d, 1H,J=8.9 Hz), 7.49 (s, 1H), 7.25 (s, 1H), 6.79 (s, 1H), 6.74 (s, 1H), 5.58(dd, 1H, J=4.4, 9.8 Hz), 5.22-5.10 (m, 4H), 4.43 (d, 1H, J=3.7 Hz),4.33-4.25 (m, 4H), 4.15-3.98 (m, 5H), 3.95-3.80 (m, 7H), 3.68-3.59 (m,1H), 3.20-3.07 (m, 2H), 2.99-2.87 (m, 2H), 2.76-2.68 (m, 2H), 1.99-1.83(m, 4H), 1.72-1.57 (m, 2H), 1.19 (d, 3H, J=6.6 Hz).

Linker-drug intermediate of ADC-52:2-((5-nitropyridin-2-yl)disulfanyl)propyl(2,5-bis((E)-3-((S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-3-oxoprop-1-en-1-yl)phenyl)carbamate(MS (ESI): 1098 [M+H]⁺) was prepared by the procedures of WO2015/023355LD-53:(S)-1-(chloromethyl)-3-((E)-3-(4-((E)-3-((S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-3-oxoprop-1-en-1-yl)-2-(2-(2-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)ethoxy)ethoxy)phenyl)acryloyl)-2,3-dihydro-1H-benzo[e]indol-5-yldihydrogen phosphate (MS (ESI): 994 [M+H]⁺) was prepared by theprocedures of WO 2015/023355

Linker-drug intermediate of ADC-54:(R)-2-((3-nitropyridin-2-yl)disulfanyl)propyl(11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(MS (ESI): 876 [M+H]⁺) was prepared by the procedures of WO 2013/055987.

Linker-drug intermediate of ADC-55:4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl)ethane-1,2-diylbis(methylcarbamate).

Following Example 3 of U.S. Pat. No. 8,389,697, to a solution ofPNU-159682 (15.3 mg, 0.02038 mmol), prepared as reported in WO1998/02446 and Example 1 of U.S. Pat. No. 8,470,984, in 3 ml of methanoland 2 ml of H₂O, a solution of NaIO₄ (5.1 mg, 0.0238 mmol) in 1 ml ofH₂O was added. The reaction mixture was stirred at room temperature for3 hours, until no starting material was detectable (TLC and HPLCanalysis). The solvents were removed under reduced pressure and thecrude red solid(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxylicacid 55a (MS (ESI): 628 [M+H]⁺) which was converted to LD-55 (MS (ESI):1355 [M+H]⁺) by the procedures of WO 2010/009124.

Linker-drug intermediate of ADC-56:(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-(2-((5-nitropyridin-2-yl)disulfanyl)ethyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide(MS (ESI): 842 [M+H]⁺) was prepared by the procedures of WO 2013/055987.

Linker-drug intermediate of ADC-57:(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-(2-((5-nitropyridin-2-yl)disulfanyl)propyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide(MS (ESI): 856 [M+H]⁺) was prepared by the procedures of U.S. Pat. No.8,389,697.

Linker-drug intermediate of ADC-58:(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-(2-methyl-2-((5-nitropyridin-2-yl)disulfanyl)propyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide(MS ((ESI): 870 [M+H]+) was prepared by the procedures of U.S. Pat. No.8,389,697.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

TABLE OF SEQUENCES SEQ ID NAME SEQUENCE NO Human CLL-1MSEEVTYADL QFQNSSEMEK IPEIGKFGEK APPAPSHVWR 1 (UniProt No. Q5QGZ9;PAALFLTLLC LLLLIGLGVL ASMFHVTLKI EMKKMNKLQN NCBI Ref.ISEELQRNIS LQLMSNMNIS NKIRNLSTTL QTIATKLCRE NP_612210.4)LYSKEQEHKC KPCPRRWIWH KDSCYFLSDD VQTWQESKMA CAAQNASLLK INNKNALEFIKSQSRSYDYW LGLSPEEDST RGMRVDNIIN SSAWVIRNAPDLNNMYCGYI NRLYVQYYHC TYKKRMICEK MANPVQLGST YFREA Human CLL-1 ECDHVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNL 2 (aa 65-265 of SEQ IDSTTLQTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFL NO: 1)SDDVQTWQESKMACAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFREA Human CLL-1 C-typeCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASLLKIN 3 lectin-like domainNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSA (CTLD)WVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEK (aa 133-250 of SEQ ID NO: 1Cyno CLL-1 MSEEVTYADLKFQNSSETEKIQEIAKFGGKAPPAPSCVWRP 4AALFLTVLCLLMLIGLGVLGSMFHITLKTAMKKMNKLQNIN EELQRNVSLQLMSNMNSSNKIRNLSTTLQTIATRLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVRTWQESRMACAAQNASLLKINNKNALEFIKS QSTSYPYWLGLSPEKDYSYGTSVDDIINSSAWVTRNASDLNNMFCGYINRIYVHYDYCI YRKKMICEKMANPVQLGFIHFREAm6E7-HVR L1 RASQSVSTSSYNYMH 5 6E7L4H1 e-HVR L1 6E7L4H1 eA54-HVR L1m6E7-HVR L2 YASNLES 6 6E7L4Hle-HVR L2 6E7L4H1 eA54-HVR L2 m6E7-HVR L3QHSWEIPLT 7 6E7L4Hle-HVR L3 6E7L4H1 eA54-HVR L3 m6E7-HVR H1 DYYMEI 86E7L4Hle-HVR H1 6E7L4H1 eA54-HVR H1 m6E7-HVR H2 RINPYNGAAFYSQNFKD 96E7L4Hle-HVR H2 m6E7-HVR H3 ERGADLEGYAMDY 10 6E7L4Hle-HVR H36E7L4HleA54-HVR H3 6E7L4HleA54-HVR RINPYAGAAFYSQNFKD 11 H2 m20B1-HVR L1SASSSISYMY 12 m20B1-HVR L2 DTSKLAS 13 m20B1-HVR L3 HQRSSWT 14m20B1-HVR H1 SYDIN 15 m20B1-HVR H2 WIYPGDGTTEYNERFKG 16 m20B1-HVR H3SYDYDYAMDY 17 m21C9-HVR L1 KASQDVSTAVA 18 21C9.L2H-HVR L1 m21C9-HVR L2SPSYRYT 19 21C9.L2H-HVR L2 m21C9-HVR L3 QQLYSTPYT 20 21C9.L2H-HVR L3m21C9-HVR H1 DYYLD 21 21C9.L2H-HVR H1 m21C9-HVR H2 RVNPYNGGTIYNQKFKG 2221C9.L2H-HVR H2 m21C9-HVR H3 DHYRYDPLLDY 23 21C9.L2H-HVR H3m28H12-HVR L1 RASQSVSSSSYSYMH 24 m28H12-HVR L2 YASNLES 25 m28H12-HVR L3QHSWEIPYT 26 m28H12-HVR H1 DTYMH 27 m28H12-HVR H2 RIDPANGDTDYDPKFQG 28m28H12-HVR H3 SGPPYYVMDY 29 m6E7 V_(L)DIVLTQSPSSLIVSLGQRATISCRASQSVSTSSYNYMHWYQQ 30KPGQPPKLLLKYASNLESGVPARFSGSGSGTDFTLNIHPVEE EDTATYYCQHSWEIPLTFGAGTKLEIKm6E7 V_(H) QVQLQQSGPELVKPGASVKISCKASGYSFTDYYMHWVKQS 31HIKSLEWIGRINPYNGAAFYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCAIERGADLEGYAMDYWGQGTSVTVS S 6E7L4Hle V_(L)DIQMTQSPSSLSASVGDRVTITCRASQSVSTSSYNYMHWYQ 32QKPGKPPKLLIKYASNLESGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQHSWEIPLTFGQGTKVEIK6E7L4Hle V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQ 33APGQGLEWIGRINPYNGAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIERGADLEGYAMDYWGQGTLVTV SS 6E7L4HleAG V_(H)EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQ 34APGQGLEWIGRINPYAGAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIERGADLEGYAMDYWGQGTLVTV SS m20B1 V_(L)DIVLTQSPAIMSASPGEKVTMTCSASSSISYMYWYQQKPGTS 35PKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAAT YYCHQRSSWTFGGGTKLEIKm20B1 V_(H) EVQLQQSGPELVKPGALVKISCKASGYTFTSYDINWLKQRP 36GQGLEWIGWIYPGDGTTEYNERFKGKATLTADKSSSTAYLQLSSLTSENSAVYFCARSYDYDYAMDYWGQGTSVTVSS m21C9 V_(L)DIQMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWFQQKP 37GQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDL AVYYCQQLYSTPYTFGGGTKLEIKm21C9 V_(H) EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYLDWVKQS 38HGESFEWIGRVNPYNGGTIYNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARDHYRYDPLLDYWGQGTTLTVSS 21C9.L2H3 V_(L)DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWFQQKPG 39KAPKLLIYSPSYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQLYSTPYTFGQGTKVEIK21C9.L2H3 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYLDWVRQ 40APGQGLEWIGRVNPYNGGTIYNQKFKGRVTLTRDTSTSTAYLELSSLRSEDTAVYYCARDHYRYDPLLDYWGQGTLVTVSS m28H12 V_(L)DIQMTQSPASLAVSLGQRATISCRASQSVSSSSYSYMHWYQ 41QKPGQPPKLLIKYASNLESGVPARFSGRGSGTDFTLNIHPVE EEDTATYYCQHSWEIPYTFGGGTRLEIKm28H12 V_(H) QVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQR 42PEQGLEWIGRIDPANGDTDYDPKFQGKATVTADTSSNTAYLQLSSLTSEDTAVYYCTISGPPYYVMDYWGQGTSVTVSS 6E7L4HleE54-HVR RINPYEGAAFYSQNFKD43 H2 6E7L4HleS54-HVR RINPYSGAAFYSQNFKD 44 H2 6E7L4HleConcensus-RINPYX₁GAAFYSQNFKD, wherein X₁ is A, E, S, or N 45 HVR H26E7L4HleConcensus- EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQ 46 HVR VHAPGQGLEWIGRINPY X₁GAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIERGADLEGYAMDYWGQGTLVTVSS, wherein X₁ is A, E, S or N6E7L4HleConcensus2- RINPYX₂GAAFYSQNFKD, wherein X₂ is A, E, or S 47HVR H2 6E7L4HleConcensus2- EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQ 48HVR VH APGQGLEWIGRINPY X₂GAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIERGADLEGYAMDYWGQGTLVTVSS, wherein X₂ is A, E, or S

What is claimed is:
 1. An isolated nucleic acid encoding an anti-CLL-1antibody, wherein the antibody comprises (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 11; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.
 2. Theisolated nucleic acid of claim 1, wherein the antibody comprises one ormore engineered free cysteine amino acid residues.
 3. The isolatednucleic acid of claim 2, wherein the one or more engineered freecysteine amino acid residues is located in the light chain.
 4. Theisolated nucleic acid of claim 3, wherein the one or more engineeredfree cysteine amino acid residues in the light chain comprises K149Caccording to Kabat numbering.
 5. The isolated nucleic acid of claim 1,wherein the antibody is a humanized antibody.
 6. The isolated nucleicacid of claim 1, wherein the antibody is an antibody fragment that bindsCLL-1.
 7. The isolated nucleic acid of claim 1, wherein the antibody isan IgG1, IgG2a, or IgG2b antibody.
 8. The isolated nucleic acid of claim1, wherein the antibody is an IgG1 antibody.
 9. The isolated nucleicacid of claim 4, wherein the antibody is an IgG1 antibody.
 10. Anisolated nucleic acid encoding an anti-CLL-1 antibody, wherein theantibody comprises (a) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:34 and (b) a light chain variableregion comprising the amino acid sequence of SEQ ID NO:32.
 11. Theisolated nucleic acid of claim 10, wherein the antibody comprises one ormore engineered free cysteine amino acid residues.
 12. The isolatednucleic acid of claim 11, wherein the one or more engineered freecysteine amino acid residues is located in the light chain.
 13. Theisolated nucleic acid of claim 12, wherein the one or more engineeredfree cysteine amino acid residues in the light chain comprises K149Caccording to Kabat numbering.
 14. The isolated nucleic acid of claim 10,wherein the antibody is a humanized antibody.
 15. The isolated nucleicacid of claim 10, wherein the antibody is an antibody fragment thatbinds CLL-1.
 16. The isolated nucleic acid of claim 10, wherein theantibody is an IgG1, IgG2a, or IgG2b antibody.
 17. The isolated nucleicacid of claim 10, wherein the antibody is an IgG1 antibody.
 18. Theisolated nucleic acid of claim 13, wherein the antibody is an IgG1antibody.
 19. An expression vector comprising the isolated nucleic acidof claim
 1. 20. An expression vector comprising the isolated nucleicacid of claim
 10. 21. An isolated host cell comprising the expressionvector of claim
 19. 22. An isolated host cell comprising the expressionvector of claim
 20. 23. An isolated host cell comprising a first nucleicacid sequence and a second nucleic acid sequence, wherein the firstnucleic acid sequence encodes a heavy chain variable region of ananti-CLL-1 antibody, wherein the heavy chain variable region comprises:(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 11; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 10; and the secondnucleic acid sequence encodes a light chain variable region of theanti-CLL-1 antibody, wherein the light chain variable region comprises:(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.
 24. The isolated hostcell of claim 23, wherein the anti-CLL-1 antibody comprises one or moreengineered free cysteine amino acid residues.
 25. The isolated host cellof claim 24, wherein the one or more engineered free cysteine amino acidresidues is located in the light chain.
 26. The isolated host cell ofclaim 25, wherein the one or more engineered free cysteine amino acidresidues in the light chain comprises K149C according to Kabatnumbering.
 27. The isolated host cell of claim 23, wherein theanti-CLL-1 antibody is a humanized antibody.
 28. The isolated host cellof claim 23, wherein the anti-CLL-1 antibody is an antibody fragmentthat binds CLL-1.
 29. The isolated host cell of claim 23, wherein theanti-CLL-1 antibody is an IgG1, IgG2a, or IgG2b antibody.
 30. Theisolated host cell of claim 23, wherein the anti-CLL-1 antibody is anIgG1 antibody.
 31. The isolated host cell of claim 26, wherein theanti-CLL-1 antibody is an IgG1 antibody.
 32. An isolated host cellcomprising a first nucleic acid sequence and a second nucleic acidsequence, wherein the first nucleic acid sequence encodes a heavy chainvariable region of an anti-CLL-1 antibody, wherein the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO:34, andthe second nucleic acid sequence encodes a light chain variable regionof the anti-CLL-1 antibody, wherein the light chain variable regioncomprises the amino acid sequence of SEQ ID NO:32.
 33. The isolated hostcell of claim 32, wherein the anti-CLL-1 antibody comprises one or moreengineered free cysteine amino acid residues.
 34. The isolated host cellof claim 33, wherein the one or more engineered free cysteine amino acidresidues is located in the light chain.
 35. The isolated host cell ofclaim 14, wherein the one or more engineered free cysteine amino acidresidues in the light chain comprises K149C according to Kabatnumbering.
 36. The isolated host cell of claim 32, wherein theanti-CLL-1 antibody is a humanized antibody.
 37. The isolated host cellof claim 32, wherein the anti-CLL-1 antibody is an antibody fragmentthat binds CLL-1.
 38. The isolated host cell of claim 32, wherein theanti-CLL-1 antibody is an IgG1, IgG2a, or IgG2b antibody.
 39. Theisolated host cell of claim 32, wherein the anti-CLL-1 antibody is anIgG1 antibody.
 40. The isolated host cell of claim 35, wherein theanti-CLL-1 antibody is an IgG1 antibody.
 41. An isolated host cell thatproduces an anti-CLL-1 antibody, wherein the antibody comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 11; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.
 42. The isolated host cell of claim41, wherein the anti-CLL-1 antibody comprises one or more engineeredfree cysteine amino acid residues.
 43. The isolated host cell of claim42, wherein the one or more engineered free cysteine amino acid residuesis located in the light chain.
 44. The isolated host cell of claim 43,wherein the one or more engineered free cysteine amino acid residues inthe light chain comprises K149C according to Kabat numbering.
 45. Theisolated host cell of claim 41, wherein the anti-CLL-1 antibody is ahumanized antibody.
 46. The isolated host cell of claim 41, wherein theanti-CLL-1 antibody is an antibody fragment that binds CLL-1.
 47. Theisolated host cell of claim 41, wherein the anti-CLL-1 antibody is anIgG1, IgG2a, or IgG2b antibody.
 48. The isolated host cell of claim 41,wherein the anti-CLL-1 antibody is an IgG1 antibody.
 49. The isolatedhost cell of claim 44, wherein the anti-CLL-1 antibody is an IgG1antibody.
 50. An isolated host cell that produces an anti-CLL-1antibody, wherein the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:34 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:32. 51.The isolated host cell of claim 50, wherein the anti-CLL-1 antibodycomprises one or more engineered free cysteine amino acid residues. 52.The isolated host cell of claim 51, wherein the one or more engineeredfree cysteine amino acid residues is located in the light chain.
 53. Theisolated host cell of claim 52, wherein the one or more engineered freecysteine amino acid residues in the light chain comprises K149Caccording to Kabat numbering.
 54. The isolated host cell of claim 50,wherein the anti-CLL-1 antibody is a humanized antibody.
 55. Theisolated host cell of claim 50, wherein the anti-CLL-1 antibody is anantibody fragment that binds CLL-1.
 56. The isolated host cell of claim50, wherein the anti-CLL-1 antibody is an IgG1, IgG2a, or IgG2bantibody.
 57. The isolated host cell of claim 50, wherein the anti-CLL-1antibody is an IgG1 antibody.
 58. The isolated host cell of claim 50,wherein the anti-CLL-1 antibody is an IgG1 antibody.
 59. The isolatedhost cell of claim 23, which is a prokaryotic host cell.
 60. Theisolated host cell of claim 23, which is a eukaryotic host cell.
 61. Theisolated host cell of claim 60, which is a Chinese hamster ovary (CHO)cell.
 62. The isolated host cell of claim 32, which is a prokaryotichost cell.
 63. The isolated host cell of claim 32, which is a eukaryotichost cell.
 64. The isolated host cell of claim 63, which is a Chinesehamster ovary (CHO) cell.
 65. The isolated host cell of claim 41, whichis a prokaryotic host cell.
 66. The isolated host cell of claim 41,which is a eukaryotic host cell.
 67. The isolated host cell of claim 66,which is a Chinese hamster ovary (CHO) cell.
 68. The isolated host cellof claim 50, which is a prokaryotic host cell.
 69. The isolated hostcell of claim 50, which is a eukaryotic host cell.
 70. The isolated hostcell of claim 69, which is a Chinese hamster ovary (CHO) cell.
 71. Amethod of producing an anti-CLL-1 antibody comprising culturing the hostcell of claim 21, so that the antibody is produced, and isolating theantibody from the culture.
 72. A method of producing an anti-CLL-1antibody comprising culturing the host cell of claim 22, so that theantibody is produced, and isolating the antibody from the culture.
 73. Amethod of producing an anti-CLL-1 antibody comprising culturing the hostcell of claim 23, so that the antibody is produced, and isolating theantibody from the culture.
 74. A method of producing an anti-CLL-1antibody comprising culturing the host cell of claim 32, so that theantibody is produced, and isolating the antibody from the culture.
 75. Amethod of producing an anti-CLL-1 antibody comprising culturing the hostcell of claim 41, so that the antibody is produced, and isolating theantibody from the culture.
 76. A method of producing an anti-CLL-1antibody comprising culturing the host cell of claim 50, so that theantibody is produced, and isolating the antibody from the culture.
 77. Amethod of producing an anti-CLL-1 antibody comprising culturing the hostcell of claim 61, so that the antibody is produced, and isolating theantibody from the culture.
 78. A method of producing an anti-CLL-1antibody comprising culturing the host cell of claim 64, so that theantibody is produced, and isolating the antibody from the culture.
 79. Amethod of producing an anti-CLL-1 antibody comprising culturing the hostcell of claim 67, so that the antibody is produced, and isolating theantibody from the culture.
 80. A method of producing an anti-CLL-1antibody comprising culturing the host cell of claim 70, so that theantibody is produced, and isolating the antibody from the culture.